The invention of the laser in 1960 gave us the ruby laser, which generally produced chaotic pulses of light. Six years later, in 1966, a concept called passive mode-locking applied to neodymium-glass lasers produced reasonably well-behaving picosecond pulses. This triggered an intense activity, with respect to developing improved laser pulse sources, measurement techniques, and application to chemistry, physics, and biology. Initially, only ∼10 –ps-long pulses at a few wavelengths were available. Nevertheless, insight into the function of complex biological systems, like photosynthetic proteins, and molecules of chemical interest was gained in very early studies. Today, both duration and color of ultrashort pulses can be tuned to almost any value. This has of course opened up possibilities to study almost any atomic, molecular, or solid-state system and any dynamic process. This review focuses on the use of laser spectroscopy to investigate light energy conversion mechanisms in both natural photosynthesis and a topical selection of novel materials for solar energy conversion. More specifically, in photosynthesis we will review light harvesting and primary electron transfer; materials for solar energy conversion that we discuss include sensitized semiconductors (dye sensitized solar cells), polymer:fullerene and polymer:polymer bulk heterojunctions (organic solar cells), organometal halide perovskites, as well as molecular and hybrid systems for production of solar fuel and valuable chemicals. All these scientific areas, and in particular photosynthesis and the solar cell materials, have been extensively studied with ultrafast spectroscopy, resulting in a vast literature; a comprehensive review of the individual materials is, therefore, not feasible, and we will limit our discussion to work that we think has been of particular importance for understanding the function of the respective systems.

With their invention, ultrashort pulse lasers have opened a wide range of new possibilities in chemical physics research and continue playing an important role. Many discoveries would not have been possible without the direct access to the timescale on which processes involving energy, electrons, atoms, and molecules occur. “Ultrashort” is of course a somewhat arbitrary concept and historically has often been used to indicate the transition from the picosecond to the sub-picosecond or femtosecond timescale. In this review, we have, however, chosen to include both the picosecond and the femtosecond timescales or, to define it more precisely, experimental work performed with both picosecond and femtosecond pulses. The ensuing dynamics following initiation with a picosecond or femtosecond pulse may start on the shortest femtosecond timescale, but carry on to much longer times, ps, ns, μs, and ms. With this definition of ultrashort pulses, our account includes the time from 1966, the year of the discovery of passive mode-locking in Nd-glass lasers and generation of the first ps pulses,1 up to the date of writing (self-locking of laser modes was in fact first demonstrated in a Q-switched ruby laser, but generated ∼1 ns pulses).2 Very recently, the generation of sub-femtosecond pulses (down to a few tens of attoseconds) has become possible and a new field, “attochemistry” is now emerging, dealing with the most fundamental atomic and molecular processes (for a review see, e.g., Ref. 3). This kind of ultrafast work is not discussed in this review.

Lasers in chemical physics research is an immense field, impossible to cover in a single review. We have, therefore, decided to first narrow the scope of our review to studies employing ultrashort pulse lasers. This is also a very large field, including areas like chemical reaction dynamics, general photophysics and photochemistry of molecular systems, energy- and electron transfer (ET), etc., showing the impact of time-resolved spectroscopy studies on chemical physics research. Therefore, we have further narrowed the review to research areas where we ourselves have been/are active, fields which we believe interest many colleagues and scientists, and which are currently also of general interest. The impact of ultrafast studies on our understanding of chemical reaction dynamics is also nicely illustrated by the Nobel Prize in chemistry 1999 awarded to Ahmed H. Zewail.4,5

The review will cover two linked areas, photosynthesis and sustainable energy materials, from the point of view of fundamental ultrafast processes. This means that we will discuss light-induced energy and ET, and related processes, in these systems. Photosynthesis research has a long history and already at an early stage of ultrafast spectroscopy, picosecond pulses were employed to study the primary ET processes in photosynthesis.6,7 It is, therefore, perhaps not surprising that photosynthetic systems and the processes therein have inspired design and study of artificial systems for harvesting and utilization of solar energy. A vast number of papers dealing with ultrafast processes in photosynthetic systems, energy materials, and devices have been published. Therefore, we will not discuss every ultrafast work on a photosynthetic preparation, or a new solar cell material. Instead, we will try to identify major steps of progress within the chosen scientific areas and discuss the results that led to new insights and understanding of function and structure.

We will start this review with a short account of the development of generation of ultrashort pulses and a few of the most common measurement techniques. This will take us from the discovery of passive mode-locking of a Nd-glass laser1 over passively and actively mode-locked dye lasers8 to Kerr lens mode-locking of the Ti:Sapphire laser. Stretching-amplification-compression of optical femtosecond pulses leading to the Nobel Prize awarded concept of chirped pulse amplification9 is a very important step forward leading to widely wavelength tunable femtosecond pulses. The x-ray free electron laser (XFEL) is the final (at this time) addition to the arsenal of ultrashort pulse lasers. Thus, the technical development over the last 30 years has taken us from ∼10 ps pulses at a few different10 wavelengths, typically 1060, 530, 355, and 266 nm, to pulses of variable duration covering the wavelengths from millimeters [terahertz (THz) pulses] to fractions of nanometers (hard x-rays). With these pulses, we are well equipped to study virtually any dynamic process in any atomic, molecular, or solid state system. We note that generation and use of attosecond pulses will not be discussed in our review, since the results of such studies still are of limited bearing on widely different chemical systems.

We will start the account of work using ultrashort pulses with studies of the primary light-induced11 processes in photosynthesis. This includes the ET processes in reaction centers, energy transfer in photosynthetic antennas, as well as photophysics and photochemistry of photosynthetic chromophores. The photosynthetic light-converting machinery consists of extensive coupled pigment systems, including several types of chromophores. To monitor the flow of energy and charge through such a system challenges most aspects of a spectroscopic measurement. Very high signal-to-noise is required in combination with very low light intensity, high temporal resolution and wide wavelength tunability. We will show how the technical progress and development in ultrafast methods have made this feasible. Ultrafast studies of carotenoid photophysics and dynamics are an example of new findings enabled by ultrashort pulses. Much of the fascinating properties of carotenoids had escaped discovery by using more conventional stationary spectroscopic methods, but application of ultrashort pulses led to major new insights into carotenoid energetics and excited state dynamics. Triggered by new experimental possibilities afforded by multidimensional spectroscopy, many photosynthetic processes and mechanisms have been reexamined recently, and also a heated debate on the role of coherent energy transfer in photosynthesis. We will summarize the various results and describe the most recent findings.

A vast number of materials for sustainable energy production have been developed over the years and studied using ultrashort pulses. Here, we will discuss the results for a few materials for capture of solar energy and conversion to either electricity in solar cells or fuel through photocatalysis. We will begin this section with a short discussion of photophysics and photochemistry of transition metal complexes since they are finding potential use both as sensitizers in solar cells and as photocatalysts. This includes excited state dynamics as it appears in the most used complexes, Ru-based and similar, as well as very recently developed Fe-based complexes with greatly extended charge-transfer (CT) state lifetimes, challenging traditional Ru-complexes as the obvious choice for photofunctional applications. We will describe the role of ultrafast studies in characterizing and understanding the excited state properties of these remarkable molecules.

Transition metal complexes have also played an important role in the development and implementation of ultrafast x-ray spectroscopy and scattering. Due to the heavy atoms with many electrons, they generate strong signals and have therefore been somewhat of a test ground for the introduction of these methods at synchrotrons and XFELs. Once optimized, the return has been rewarding and the species and state selectivity of x-ray methods have delivered new knowledge on for instance relaxation pathways and state characteristics, not obtainable with optical methods. We will show how this was possible.

The dye-sensitized solar cell (DSC), with an active material consisting of a nanocrystalline metal oxide thin film sensitized to visible light by a dye, was invented in 1991 by Grätzel and O'Regan.12 Since then, thousands of dye–semiconductor combinations with various electrolyte systems have been developed and led to a slow but steady improvement of light to charge conversion efficiency, from ∼7% to today's ∼15%.13 We will discuss light-induced dye-to-semiconductor electron injection, recombination as well as charge carrier mobility and transport, key processes for understanding the function of a DSC that start on the ultrafast timescale and may proceed into the nano-, micro-, and millisecond time domains.

The so-called bulk heterojunction (BHJ) concept (see, e.g., Ref. 14) is the most successful design of a fully organic solar cell material. From a superficial view, it somewhat resembles the organization of a DSC material; it has a light-absorbing material, a conjugated polymer, acting as electron donor and a fullerene or another polymer as electron acceptor, all blended into nanodomains, or down to the molecular level. Also here, charge generation, recombination, and transport are the fundamental processes underlying the function of a solar cell. DSC and BHJ materials with their nanostructured light-harvesting and electron donor/acceptor organization have often been compared to photosynthetic systems and inspiration for their design borrowed from the latter.15,16

As we will see from our presentation below of DSC and BHJ dynamics, decisive functional steps occur on the ultrafast, often sub-100 fs, timescale, frequently in competition with other less productive relaxation processes. Ultrafast methods are, therefore, powerful tools to monitor reaction pathways and identify bottlenecks toward optimizing light-to-charge conversion efficiency of the materials. Our review will illustrate this with several examples.

Organometal halide perovskite (OMHP) materials are the most recent addition to the family of photovoltaic (and luminescent!) materials, which in just a few years have risen to a serious competitor to more established inorganic semiconductor materials. The interplay between electronic and structural degrees of freedom in OMHPs results in unusually rich and interesting dynamics, which the wide variety of ultrafast tools is very well suited to disentangle. We will discuss the results illustrating the complex carrier and structural dynamics of the perovskites, providing some answers to their remarkable properties.

Solar energy, like wind energy, is intermittent and its optimal utilization therefore requires storage possibilities. This can be, for example, achieved with solar cell driven electrolysis of water, but a more direct way of achieving it would be through photocatalytic processes emulating photosynthetic water oxidation and production of molecular hydrogen, or some other fuel. Development of photocatalysts and studies of the light-induced processes have recently appeared as a new research field on its own. Here, we will summarize some of the ultrafast spectroscopy work aimed at characterizing initial photoinduced energy- and ET processes in a few model catalysts.

This is not a review of the individual photosynthetic systems or materials designed for harnessing solar energy by conversion to electrons (i.e., solar cell materials) or energy-rich molecules (e.g., solar fuel). Instead, the main aim with this review is to illustrate how ultrafast spectroscopy, from the early days of its conception until today, has contributed to our understanding of the function of nature's most important process, photosynthesis, and novel solar energy materials. In our discussion of the various photosynthetic and material systems, we have, therefore, focused on the achievements, and future perspectives will be only briefly discussed when ultrafast spectroscopy clearly could shed new light on unresolved issues. In any molecular or solid state material, in general, whose function emanates from interaction with electromagnetic radiation by absorption of photons, the functional processes compete with radiative or radiationless decay of excited states. To be efficient, the functional processes have to outcompete these “non-productive” processes that often proceed on the fs to ns timescale. This means that in order to get insights into function for any such system, it is necessary to study and understand the various processes that start in the excited state and eventually lead to a product or some nonproductive state (e.g., the ground state). Thus, ultrafast spectroscopy, in its many incarnations, has been used to disentangle the processes leading to all possible final states.

The ultimate timescale of processes involving the smallest chemical entities, electrons, atoms, and molecules, is set to femtoseconds and picoseconds by the smallness of these units and their characteristic motions. The direct access to this, the atomic and molecular timescale, has been approached through a stepwise technical development for generation of short pulses of electromagnetic radiation (light from now on), over a period of 70 years or more. Strategies relying on the generation of short bursts of other form of energy, i.e., electrons17,18 or heat,19 or high frequency modulation of continuous light,20 have also been developed, but the concept involving light pulses has turned out to be the winning approach with detection techniques relying on the principle of photography.

This development started with the introduction of the flash photolysis technique using millisecond flashlamp pulses in the late 1940s and early 1950s.21 The flash of light initiated a chemical reaction in the studied sample and the progress of the process was monitored by measuring changes in absorption of a continuous light beam detected with a photomultiplier and time resolved with for instance an oscilloscope. Much later with the use of increasingly shorter light pulses, the technique has been named pump-and-probe and many different spectroscopic techniques have been used for detection. In such an experiment, both pump and probe are short pulses of light, and time resolution is achieved by a variable time delay between pump and probe, realized with an optical delay line; the probe pulse simply travels a variably longer distance than the pump pulse. This means that no fast detection equipment is needed—an optical path length of 0.3 mm corresponds to 1 ps and time delays down to sub-fs times can easily be achieved. Early versions of pump-probe measurements relied on lasers with low pulse repetition rate, or even single pulse lasers. Modern pulsed lasers have kHz to MHz pulse repetition rates, which implies that a measurement in effect is repeated at this frequency leading to powerful signal averaging. Following this section on short pulse generation, we will describe a few of the most used detection techniques.

With the advent of the laser and giant pulse formation, or Q-switching, in the early 1960s22,23 the microsecond and nanosecond timescales were opened for investigation. Only a few years later, in 1966 passive mode-locking of a solid state Nd3+-glass laser, with the help of a saturable absorber dye, produced ∼10 ps pulses;24 similar mode-locking of the related Nd3+-YAG laser, having somewhat narrower spectral bandwidth of the active laser medium, resulted in ∼30 ps pulses. This development paved the way to the new field of picosecond spectroscopy. These solid state lasers were pumped by flash lamps at low repetition rates, typically a few Hz or lower, and the resulting pulses were of high energy. This often caused unwanted non-linear effects in the studied samples, which led to distorted spectral and kinetic response. The low repetition rate and large pulse-to-pulse intensity variation, in addition, made signal averaging of measured responses practically impossible.

Mode-locked dye lasers, which typically were pumped by a continuous or mode-locked (MHz pulse repetition rate) ion laser produced intensity-stable low-energy pulses at MHz repetition rates. This made lock-in detection possible, which drastically improved the sensitivity of measurements. Pumped by a CW ion laser, the dye laser was passively mode-locked with a saturable dye solution.25,26 In a linear cavity configuration pulses as short as 0.3 ps were reported,27,28 whereas colliding pulse mode-locking in a circular cavity configuration generated much shorter, less than 100 fs pulses,29 and with dispersion compensation pulses as short as 27 fs could be generated.30 The necessity to match absorption properties of lasing and mode-locking dyes for optimum pulse width limits the wavelength tunability to typically 10–20 nm; wavelengths within a range of a few tens of nanometers around 600 nm were therefore the most used.

Active mode-locking, or synchronously pumping with high repetition rate (typically ∼80 MHz) mode-locked ∼100 ps ion laser pulses, was the remedy to the limited tuning range. Virtually all laser dyes absorbing the ion laser wavelength could be used with synchronous pumping, resulting in ps pulses of a few ps duration from the near infrared to the green colors in the visible.8 Synchronous pumping of the dye laser did, however, not generate short sub-picosecond pulses as achieved with passive mode-locking.

The oscillator outputs of high repetition rate (typically ∼80 MHz) nanojoule pulses were often used directly in experiments, and sometimes at somewhat reduced repetition rates by Bragg cell cavity dumping or Pockels cell pulse picking. The high pulse-to-pulse amplitude and pulse width stability enabled lock-in amplifier detection and high signal-to-noise ratio measurements. In contrast to the early low repetition rate, high pulse energy, picosecond lasers this now allowed efficient signal averaging and measurements at very low excitation pulse energy, avoiding unwanted non-linear effects that distorted the response of the system under investigation. Measurements with these high rep-rate, low pulse energy lasers were generally one-color measurements, i.e., pump and probe were of the same wavelength. Dual-color pump-probe measurements with limited choices of pump and probe wavelength combinations could, however, be realized with multiple dye laser oscillators, pumped by the same pump laser. Since both laser oscillators were pumped by the same laser, pulses from the two dye lasers were perfectly synchronized.31 

The invention of the Ti:sapphire laser in 1982 (Ref. 32) and its Kerr-lens mode-locking33 combined with chirped pulse amplification9 implied nothing less than a revolution for ultrafast science. A Ti:sapphire oscillator typically produced ∼650–1000 nm sub-100-fs transform-limited pulses, which could be stretched to durations such that amplification to high power still was below damage threshold of the amplifier material. With the help of prisms or gratings, the long amplified pulses could then be compressed back to the original transform-limited duration. The peak power of the amplified and compressed pulses is sufficiently high to facilitate frequency shifting over wide ranges with the help of a range of non-linear optical effects, e.g., harmonic generation (e.g., second-, third-, and fourth-harmonic), optical parametric generation and amplification (OPG/OPA), and white light continuum generation with high conversion efficiency. The result is sub-100-fs pulses of almost any wavelength from the far infrared to the ultraviolet that allow for studies of most atomic, molecular and material systems. Single-cycle THz34 pulses of ps duration, generated through, for instance, optical rectification35 and sub-fs soft x-ray pulses generated by high harmonics generation36,37 extend this range further.

Synchrotrons and hard x-ray pulses opened this energy range of the electromagnetic spectrum for analysis in time-resolved studies, albeit with a time resolution limited by the ∼100 ps synchrotron pulses.38,39 Pulse slicing40 extracted a shorter temporal slice of the synchrotron pulse and enabled in principle measurements with x-ray probing with few-ps resolution. The low number of x-ray photons in an x-ray slice and the required very long measurement times, however, prevented the widespread use of this technique.

With the advent of the XFEL, this has all changed. With several facilities in operation worldwide, ultrafast x-ray spectroscopy and scattering (see, e.g., Ref. 41) measurements with sub-ps temporal resolution and probing over a wide range of x-ray energies are now becoming routine. The very short, often ∼10 fs, duration of the x-ray pulse implies that it is no longer the x-ray pulse itself that sets the limits for time resolution, but rather the duration of the optical laser pulse used for excitation and the synchronization between optical and x-ray pulses. Increasingly more precise timing tools to compensate for the jitter between optical and x-ray pulses pushes time resolution of experiments to the limits defined by the cross correlation between exciting and probing pulses.42 

Dynamic chemical responses can be monitored and time resolved with the help of many different methods, absorption or emission of light, elastic or inelastic scattering, non-linear responses, etc. Absorption or emission of light is perhaps the conceptually most straightforward methods and depending on which part of the electromagnetic spectrum is used, they form a variety of different spectroscopies—starting from the low energy end of the spectrum, e.g., THz spectroscopy, IR spectroscopy, uv/vis spectroscopy, x-ray spectroscopy. In all methods where detection is realized with the help of a probe beam (e.g., absorption and scattering processes), time resolving the sample response relies on the pump-probe concept and a time delay between pump and probe pulses. Like all pump-probe techniques, transient absorption is a third-order nonlinear technique, meaning that during the measurement the sample interacts with three electric field instances. Time resolving of sample luminescence response can be achieved both electronically (e.g., with a streak camera) and based on the pump-probe concept in a gating experiment (e.g., fluorescence upconversion or Kerr gate detection). Detection in the early picosecond experiments was performed by monitoring changes in absorption (or transmission) of visible probe light, or time resolving sample luminescence, and still today these are extensively used methods. Below, we will describe these “standard” methods, as well as a few somewhat more elaborate methods.

1. Transient absorption

To illustrate both the principle of a transient absorption measurement and the impressive improvements in measurement quality and accuracy enabled by technical progress over several decades, we show how a typical measurement was performed when one of us had the privilege to work as a Ph.D. student at Bell Laboratories in 1975. The experiment we have chosen was to characterize the intersystem crossing from the S1 to the T1 state in an aromatic molecule, acridine, by time resolving the process and to measure the absorption spectrum of the T1 state.43 

The layout of the laser and detection system used for these measurements is illustrated in Fig. 1. The laser was a passively mode-locked Nd3+-glass laser amplified in two stages. The oscillator generated a microsecond-long pulse train of approximately hundred 10 ps pulses spaced by ∼10 ns, but only pulses in the beginning of the train were of sufficient quality to be used in the measurements. One single ps pulse was, therefore, extracted from the train by a Pockels' cell triggered by the high-voltage pulse generated by a laser initiated breakdown in a “spark gap.” The pulse repetition rate of the laser was one “shot” every ∼10 min—or, every time the flashlamp and capacitor bank used for firing the lamp discharge had recovered from the previous shot. This means that a measurement of a kinetic trace or a spectrum had to be completed with one single pulse, since the low reproducibility of pulses made averaging meaningless. How do you do that? Probe light was, like often still today, a white light continuum, but with one big difference. White light continuum generated with picosecond pulses of insufficient peak power to saturate the non-linear effects is of poor quality, with large intensity and spectral variations from pulse to pulse. “Scanning” time delay between pump and probe pulses was performed by sending the white light continuum through an “echelon” (item 11 in Fig. 1)—a stair-like piece of quartz breaking up the continuum pulse into several segments separated in time by the difference in thickness of the quartz stairs. As illustrated in Fig. 2, ten time points or so could be obtained covering a time window of a few-100 ps. A sample and reference beam for measuring I and I0 was absolutely necessary to compensate for the spatial intensity variation of the continuum after passing the echelon. Both I and I0 beams were sent through a spectrometer and registered by an optical multichannel analyzer. Although no error bars are indicated in the kinetic trace of Fig. 2, the accuracy was probably no better than ± 0.1 ΔOD unit.

Since then, progress in ultrashort pulse generation and detection technology over several decades has made transient absorption measurements a versatile and powerful technique. Key elements of this development are femtosecond pulses of almost any wavelength at high and variable repetition rates with high pulse-to-pulse intensity and pulse width stability. This enables studies of a broad variety of atoms, molecules, materials, and processes with detection of very weak signals with ΔODs on the order of 10−5–10−6.44,45 Complete transient absorption spectroscopy systems are available for purchase since a couple of decades, which further spreads the use of the technique.

Transient THz spectroscopy

Transient absorption spectroscopy is generally performed with probe pulses in the IR-UV spectral regions, but such probes are not always best suited for unraveling photoexcited dynamics. For example, the function of, e.g., photovoltaic materials rely on the generation of mobile charges that can be harvested as a photocurrent (PC). Conventional transient absorption spectroscopy is insensitive to the dynamics (mobility) of free charges. However, to directly access the behavior of charge carriers on the ultrafast timescale, time-resolved terahertz (THz) spectroscopy (TRTS) is a powerful tool. Upon pulsed (∼80–100 fs) light excitation with a photon energy above the bandgap of a molecule or solid state material, charged species, either loosely or tightly bound, are generated. This results in a change of photoconductivity (Δσ) in the material, which will modulate the characteristics of pulsed THz radiation used as a probe. This change in Δσ can be calculated using the following relation:46 

Δσnexce0=φμe+μh=ΔEexcωEgsωϵ0cFe011eαL,

where nexc is charge density, e0 is the elementary charge, φ is photon-to-charge conversion quantum yield, μe and μh are the electron and hole mobility, respectively, ΔEexc and Egs are the THz electric field transmitted through the sample with and without light excitation, respectively, ε0 is permittivity of vacuum, c is velocity of light, F is the fluence of the excitation light in ph/cm2, α is the absorption coefficient, and L—the thickness of the sample. The quantity that can be obtained from this equation is the total carrier mobility in cm2/V s. As shown, the measured change in photoconductivity, Δσ, is a product of quantum yield and mobility, meaning that in order to obtain Δσ, the photogenerated species should be both charged and mobile. A tightly bound molecular exciton, which may be created by light excitation, will not be detected since by definition it has no net charge. In the same manner, if the pump pulse creates ions, whose mobility is very low, the sensitivity of a particular measurement may not be sufficient to detect them. The temporal evolution of the charge population and mobility defines the shape of the THz transient photoconductivity kinetics. On the one hand, a rise in photoconductivity kinetics reflects generation of charged species and/or increase in mobility of the charges. On the other hand, a decay represents the decrease in the mobility (e.g., due to relaxation or trapping) and/or disappearance of charge carriers either by recombination, or injection to a low-mobility acceptor material.

Pulsed THz radiation is conveniently generated through an optical rectification process by pumping a ZnTe crystal with 800 nm, ∼100 fs, and 100 μJ pulses.47 Another ZnTe crystal is used for detection by spatially and temporally overlapping the pulsed THz radiation with 800 nm gating pulses in a process known as electro-optical sampling. The transient THz photoconductivity kinetics is collected by fixing the delay of the 800 nm gating pulses at the peak of the THz electric field and scanning the pump-probe delay within a desired time interval, typically up to 1 ns. To obtain a THz photoconductivity spectrum, the pump-probe delay is fixed at a desired pump-probe delay while the delay of the 800 nm gating pulses is swept to map the THz electric field. The measured Δσ as a function of THz frequency represents the photoconductivity spectrum, and from the shape of this spectrum carrier scattering time and carrier localization can be determined.46,48 It should be noted that at the earliest timescale, φ is often assumed to be unity, while at longer times this represents the change in charge population at a particular time. φ close to 1 means that all absorbed photons are converted to mobile charges. However, since accurate measurement of φ is generally difficult, this assumption means that reported mobility values are lower limits and can in reality be considerably higher.

2. Time-resolved fluorescence

For time and spectrally resolving of sample luminescence four different methods are in frequent use, two based on fast electronic detection [time correlated single photon counting (TCSPC) and streak camera] and two relying on the pump-and-probe principle through a gating process (fluorescence Kerr gate and fluorescence upconversion). All four techniques are described and discussed in the excellent book by Lakowicz,20 so only a very brief description is given here.

Both TCSPC20 and streak camera measurements49 are very well-established techniques with complete measurement equipment and devices available from several commercial vendors. TCSPC is designed to detect single fluorescence photons from a periodic signal excited by a pulsed laser with high repetition rate. A time-to-amplitude converter converts the short time period between the excitation laser pulse (start pulse) and the first detected fluorescence photon (the stop pulse) to a voltage, which is digitized in an analog-to-digital converter and stored in a multichannel analyzer. By repeating this many, many times a histogram of detected fluorescence photons at a particular arrival time representing the fluorescence decay is constructed. Since single photons are detected, this is a very sensitive method and the shortest decay times that can be retrieved with this method is 10–20 ps. TCSPC measures fluorescence decays at a single wavelength. If the time dependence of a fluorescence spectrum is needed, many time traces at different wavelengths have to be measured and combined into a spectrum.

A streak camera49 is a device featuring two-dimensional detection, meaning that the full temporal evolution of a fluorescence spectrum can be directly measured. Time resolution in a streak camera is achieved by converting time into a distance on a detector phosphor. Fluorescence photons generate photoelectrons at a photocathode, and these electrons are deflected by a high-voltage sweep perpendicular to their direction of propagation in a streak tube. On the detector phosphor, they form a two-dimensional streak image where one axis represents time and the perpendicular axis—wavelength. Streak tubes with sensitivity from the x-rays to the infrared and featuring single photon detection can be obtained. Hamamatsu, one manufacturer of streak cameras, states that their single scan camera for single events has a time resolution of 100 fs, while their synchroscan camera for repetitive events has a time resolution of  ≤800 fs.

In both the Kerr gate50 and upconversion51,52 fluorescence detection time resolution is achieved with the help of an “optical shutter” that cuts temporal slices out of the fluorescence decay. The gate timing is controlled by a delay stage, which sets the time difference between fluorescence excitation pulse and gate pulse. Sweeping the delay and measuring the intensity of the fluorescence slices yield the decay profile. The Kerr gate consists of a liquid (e.g., CS2) or solid material (e.g., thin glass slide) placed between two crossed polarizers. In the non-active state, the Kerr medium is isotropic and no fluorescence is transmitted through the gate. Applying an optical gate pulse induces birefringence in the Kerr medium, which rotates the polarization of transmitted fluorescence and causes a portion of the fluorescence to pass through the second polarizer. The duration of the light burst transmitted through the Kerr gate, and thus, the time resolution is determined by the duration of the gate pulse, the relaxation time of the induced birefringence and the thickness of the Kerr medium. In the original setup developed by Duguay,50 the fluorescence spectrum was integrated, but since the Kerr gate has no wavelength discriminating components, it is in principle a broad-band method allowing broad spectrum detection. This was later implemented by Schmidt and co-workers in a modified setup.53 With 100-fs gate pulses, a Kerr medium with short relaxation time can be chosen, such that time resolution is mainly limited by the duration of the gate pulse.

In a fluorescence upconversion experiment,51,52 the gate medium is a non-linear crystal and the gating mechanism is sum frequency generation between the fluorescence light and the gating pulse. Other nonlinear frequency conversion modalities, such as difference frequency generation, can be also used for the gating. An upconverted signal is only present when both fluorescence and gate pulse overlap temporarily and spatially in the non-linear crystal and frequency upconverted radiation (νfluorescence + νgate) is generated. The time resolution of an upconversion experiment is mainly determined by the duration of the gate pulse and thickness of the non-linear crystal. As originally developed, fluorescence upconversion was a single-color technique—the phase matching angle of the non-linear crystal had to be optimized for each fluorescence wavelength.51 Later, a broadband version of the method was developed, with a judicious choice of non-linear crystal and phase matching conditions.54 

3. Multipulse techniques

a. Femtosecond stimulated Raman spectroscopy

To obtain dynamical structural information about molecules and different materials, that is, ultrafast changes of structural configurations following light excitation, probing vibrational transitions after the excitation pulse is desirable, as different structural conformations give rise to different vibrational responses. There are several time-resolved methods, including femtosecond stimulated Raman spectroscopy (FSRS), pump-degenerate four wave mixing (DFWM), and visible-IR two-dimensional spectroscopy, etc., capable of such investigations. We start with the FSRS technique, which is a fifth-order (in interaction with the electromagnetic fields) nonlinear technique that probes vibrations on the excited electronic states.

Time-resolved Raman spectroscopy was first implemented in the nanosecond and then picosecond time domains. In 2000, Yoshizawa and Kurosawa used a trick of separating the narrowband Raman pump pulse and the broadband Raman probe pulse in FSRS to push time resolution into femtoseconds.55 The typical implementation of FSRS involves three pulses: the first actinic pulse, usually in the UV or Vis range, is used to promote a substantial part of the sample to the excited state. Then after some delay time, simultaneously, the narrowband Raman pump pulse (1–4 ps long) and broadband Raman probe pulse (<30 fs) are sent, and changes in spectral amplitude of the probe pulse are detected.56 Positive or negative Raman lines are detected on top of the smooth probe spectrum with the spectral resolution of 10 cm−1. Since the free induction decay signal of the superposition of vibrational states is measured, which takes a picosecond or even longer to develop, the “real” resolution of the experiment is not femtosecond. However, one captures the system state at the moment of the arrival of the femtosecond probe—in a sense at the moment when free induction decay is starting—so typically it is said that, similar to pump-probe, the time resolution of the FSRS experiment depends on the length of the actinic and probe pulses. The signal is corrected for probe fluctuations and background. For this, the Raman probe spectral profile, but also the solvent Raman signals have to be measured and subtracted. Sometimes the ground state signal of the system is also subtracted to leave only the excited state contributions. Both non-resonant and resonant Raman pump pulses have been used in the experiments,57–59 and the two approaches have their own advantages and disadvantages. For example, changing Raman line shapes were investigated, by tuning Raman pump frequency across the excited state resonance.58 

b. Pump degenerate four wave mixing

Another fifth-order non-linear technique sensitive to vibrational signals is pump-DFWM.60,61 In contrast to FSRS, this is a fully time domain technique. The first step in the experiments is the same—the strong actinic pump pulse is used to excite the sample to an excited electronic state. This excitation is followed by a sequence of three pulses constituting the four wave mixing arrangement. The three pulses are pump, Stokes, and probe. The pump and Stokes pulses prepare a vibrational superposition (a wavepacket) in the excited state (vibrational “coherence” signal), which is then probed by the delayed probe pulse. The usually used homodyne detection of the oscillating signals requires some monotonously decaying signal, which is conveniently provided by the population dynamics signal. The vibrational spectrum in the excited state is obtained by Fourier transforming the signal dependence on the delay time between the pump/Stokes pulses and the probe. The time delay, corresponding to the delay in pump probe spectroscopy, is the delay between actinic and pump/Stokes pulses.61 

Both FSRS and pump-DFWM provide complementary information to the more common pump-probe and time-resolved fluorescence techniques, because they directly probe vibrational transitions in the excited state. However, generally a large variety of signals can be measured in fifth-order techniques and great care has to be exercised to distinguish the signals of interest. Consequently, interpretation of the signals is often not straightforward.

c. 2D electronic spectroscopy

Inspired by the tremendous success of multidimensional NMR spectroscopies, the analogous experiments were implemented in both the IR62 and visible wavelength ranges,63,64 as well as the combination of the two.65 Most commonly, the results obtained with these techniques are presented in two dimensional graphs, involving excitation and detection frequencies, and therefore, all nonlinear spectroscopy methods listed here are called two-dimensional spectroscopies. The main idea here is to measure correlations between initially excited transitions and transitions probed after the delay time.66 The evolution of these correlations is then represented in the 2D maps. Just like pump-probe spectroscopy, 2D spectroscopy is a third order non-linear spectroscopy technique. This four-wave-mixing experiment relies on the assumption that the sample interacts with three weak exciting fields (pulses), whereupon transient polarization is created in the sample, which is then emitted as electromagnetic radiation and measured using heterodyne detection. Two main differences and at the same time advantages can be listed when compared to conventional pump-probe spectroscopy. First, full signal information, i.e., amplitude and phase, is measured in 2D spectroscopies, and second, excitation frequency resolution is achieved.

As 2D spectroscopies enjoy widespread popularity, multiple realizations in different spectral ranges can be found using various geometries, where phase matching conditions and/or pulse manipulation with pulse shapers are implemented. As a typical representation, here we focus on 2D electronic spectroscopy (2DES) in a non-collinear configuration,64,67 where four pulses are used in the experiment. The first two pulses with a coherence delay introduced between them with interferometric accuracy, t1, correspond to the single pump pulse in pump-probe spectroscopy. The second delay, t2, between the second and the third pulse, called population delay, corresponds to the time delay in pump-probe experiments. Finally, a signal is emitted following the third pulse at a delay t3. The fourth so-called local oscillator pulse is phase locked to and delayed in respect to the third pulse. This is necessary for heterodyne detection, where an interferogram between the signal and local oscillator is measured in a spectrometer. Usually, four pulses with wave vectors k1 k2, k3, and kLO are geometrically arranged in the corners of a square—facilitating measurements of only the phase matched signals in the kS = −k1 + k2 + k3 direction, which coincides with the direction of the local oscillator pulse kLO. In this way, the number of signals detected in the experiment is highly reduced, in turn aiding interpretation of measured data.

The signal is extracted from measured interferograms by linear spectral interferometry techniques using Fourier filtering68 and has information on both amplitude and phase. Thus, full information is obtained on the third order polarization created in the sample, in contrast to pump- probe measurements, where only amplitude is measured. The extracted signal has detection frequency information, whereas a Fourier transform over the coherence time completes the procedure and yields the excitation frequency information.

The spectral information that can be obtained about the system under investigation is limited by the laser spectrum used in the experiment. Therefore, spectrally broadband and ultrashort laser pulses are highly preferable, and thus, 10–15 fs pulses are often used. Consequently, the time resolution of the 2DES experiment is in the range of 20 fs. At the first glance, it seems paradoxical that high time and spectral resolutions are simultaneously achieved, both for excitation and detection frequencies. This could be interpreted as contradicting the time-frequency uncertainty principle regarding the laser pulses used in the experiment, which indeed limits spectral resolution of pump-probe experiments. This apparent paradox is overcome, because excitation frequency resolution is not achieved in a single experiment, but reconstructed with the help of a Fourier transform from multiple experiments where time delay between the first two pulses is scanned.

As already mentioned, the main advantage of 2DES is excitation frequency resolution, which simply allows one to “see the full picture” in a single experiment, whereas in more limited experiments some important details could be missed. Additionally, 2DES can further help disentangling several types of congested signals. This includes the possibility of separating rephasing and non-rephasing signals, which are inseparably mixed in pump-probe measurements. This separation is of great help when disentangling oscillating (or coherence) signals. Furthermore, phase information present in 2DES experiments allows for identifying the “direction” of phase evolution of coherence signals. An everyday analogy for this is the propagation direction of ripples caused by a stone thrown in a pond. In other words, whereas in 2DES these signals evolve on the full complex plane, only the real part projection is seen in pump-probe measurements. Finally, 2DES facilitates manipulation of all four “waves” in the four-wave-mixing experiment. By controlling polarization of the three interacting pulses and filtering the polarization component in the detected signal, a wide range of anisotropy-type of experiments can be carried out. An example of polarization-controlled 2DES experiment, beyond that available in pump probe, is (45°, −45°, 90°, 0°),69–71 where the angles in parenthesis indicate the relative orientation of the linear pulse polarizations and filtered polarization of the signal. This polarization sequence, called double-crossed polarization, allows for suppressing all signals except coherences, which are excited via interaction with the transitions having non-parallel dipole moments. This implies that only purely electronic coherences, or vibrational coherences excited via vibronically mixed states are selected. This is arguably the only method, which allows for direct detection of vibronic mixing in molecules72 and molecular complexes.71,73,74

All photosynthetic processes in nature occur under ambient light, while ultrafast spectroscopy makes use of laser pulses, which are qualitatively very different from sunlight. The laser light used in ultrafast spectroscopy is coherent, usually highly polarized and has a large number of photons packed into a very short duration in time. Laser light also has a frequency comb spectrum making it very different from sunlight experienced by photosynthetic organisms in their natural environment. Thus, one fundamental question is how the large qualitative difference between experimental (ultrashort laser pulses) and natural (sunlight) light sources may affect the interpretation of experimental results toward understanding of photosynthetic functions (and human-made solar energy conversion).

In photosynthesis, the light intensity is traditionally specified in micro-Einsteins per square meter per second (μE·m−2·s−1) and the light intensity corresponding to sunlight vary between 200 and 2000 μE·m−2·s−1.75 Since the unit of Einstein denotes one mole (6.023 × 1023) of photons, this number can be recalculated to the photon flux of approximately 1020–1021 photons·m−2·s−1. Then, for chlorophyll which has an absorption cross section of σ ∼ 1 Å2,75 this translates to 1–10 absorbed photons per chlorophyll per second. Thus, a chlorophyll molecule even at full sunlight is promoted to its excited state approximately once per 100 ms. This is in vast contrast to photon excitation densities typically used in ultrafast spectroscopy. The excitation intensities used in a typical transient absorption experiment employing a 1 kHz pulse repetition rate are in the range of 1013–1014 photons cm−2·pulse−1, which, with pulse durations of 30–100 fs, corresponds to a photon flux reaching values as high as 1030 photons·m−2·s−1 during the pulse duration, thus many orders of magnitude higher than full sunlight. Of course, since for 1 kHz repetition rate we have only 1000 such intense short pulses per second, the total number of photons hitting the sample within one second is comparable to that under full sunlight. However, the photon flux within the ultrashort pulse is enormous and incomparable with natural light conditions.

The key question in the field of ultrafast spectroscopy of photosynthetic, or other biological systems, is how these large photon fluxes may affect the outcome (and interpretation) of experiments. It is important to realize that if a chlorophyll molecule under full sunlight is excited approximately once per 0.1 s, it means that even for the largest antenna systems at a given time moment there is never more than one excitation per whole antenna (this holds also for other photosynthetic pigments, bilins, and carotenoids since their molar absorption coefficients are comparable). If we assume a model antenna system containing a connected array of 100 pigments [e.g., the photosystem I (PSI) core, see Sec. III E 2 a], it will be excited by sunlight with a rate of about one excitation per millisecond. Thus, if the trapping time of such a system is ∼100 ps, most of the time there is actually no excitation in the system as the trapping time is seven orders of magnitude faster than the rate by which the system is excited.

This situation, however, differs for photon fluxes used in ultrashort laser pulses. We again assume the same model antenna of 100 pigments, each having a molar absorption coefficient approximately equal to that of Chl a, which results in a total cross section of the whole antenna of σ ∼ 4 × 10−14 cm2. Then, if the intensity of the laser pulse (I) is given in photons cm−2·pulse−1, the average number of pigments excited simultaneously by such a pulse is x=Iσ. Using Poisson distribution, we can calculate the probability of simultaneous excitation of n pigments within our model antenna,

Pn=xnexn!.

Then, P0 is the probability that no pigment is excited, P1 gives the probability of excitation of one pigment in our model antenna and so on.76 These probabilities and average numbers of simultaneously excited pigments in our model antenna are given in Table I for some typical excitation intensities used in ultrafast experiments.

The table shows that for such a model antenna, using intensities >1013 photons pulse−1 cm−2 inevitably leads to a non-negligible probability of exciting more than one pigment within the antenna. If these two pigments are connected via some energy transfer pathway and their excited state lifetime in the antenna is longer than the time it takes for the two pigments to meet, it will lead to annihilation resulting in excitation intensity dependent dynamics (Sec. III). It is extremely important to consider this effect especially for large antenna systems such as chlorosomes (Sec. III C 2), for which excitation intensities as low as ∼1011 photons pulse−1 cm−2 are required to obtain annihilation-free dynamics.77 The presence of multiple excitations within a connected array of pigments may also lead to population of higher excited states that may initiate photophysics that is not related to the initially excited state, complicating interpretation of data.78 

While the problems related to high photon fluxes resulting in annihilation are well understood and can be taken into account in data analysis,79,80 the issues related to the fundamental difference between incoherent sunlight and coherent ultrashort laser pulses are much more difficult to evaluate. The coherent vibrations induced in photosynthetic proteins after excitation by sub-100 fs pulses were reported already in 1991 in photosynthetic reaction centers81 and in 1994 for a light-harvesting antenna.82 However, the role of coherence in photosynthesis has become a highly debated topic more than a decade later, and 2DES experiments played a central role (Sec. II B 3 c). The first 2DES experiments on photosynthetic systems suggested the key role of coherences for the efficiency of energy transfer, leading to the advent of the field of “quantum biology.”83 These and subsequent studies reporting the presence of coherences have led to a hypothesis that these quantum coherences are nature's tool to efficiently direct energy transfer through light-harvesting systems. Nearly fifteen years of discussions have eventually provided a better understanding of these phenomena, concluding that most of these long-lived coherences have their origin in collective vibrations generated by impulsive excitation by coherent ultrashort pulses.84 A more detailed description of these phenomena is provided in Sec. III H.

Here, we would like to discuss an even more fundamental issue. All experiments addressing the ultrafast dynamics of photosynthetic systems are based on laser-induced coherent excitations, while under natural conditions photosynthetic systems are excited by incoherent sunlight. The relation between experimental observations and processes occurring in nature remains a matter of considerable debate as these two situations inevitably lead to a different response of a studied system.85 The excitation with an ultrashort laser pulse represents a well-defined coherent field, having a coherence time much longer than the lifetime of the studied system, inducing a time-dependent perturbation. In contrast, sunlight is incoherent (the coherence time of just a few femtoseconds) and thus characterized as an ensemble of fields described by their statistical properties.86 Thus, under incoherent sunlight the system is in a non-equilibrium steady state, free from any time-dependent coherences. Yet, static coherences, which may result from system-bath couplings, may occur in such a steady state regime.87 In this non-equilibrium steady state, also the meaning of decay rates obtained from the time evolution of a system perturbed by an ultrashort pulse was suggested to be different.85 

Despite the concerns given above, there is no doubt that the experiments using ultrashort laser pulses, which are at the focus of this review, provide a wealth of information about the studied system. They give accurate information about the electronic structure, interaction of the pigments with their environment and, via measuring the decay rates, reveal the pathways and efficiencies of energy flow within the system. However, the exact description of how a light-harvesting system will behave under incoherent sunlight, which would correspond to a chlorophyll molecule absorbing approximately one photon per second, is still not completely clear. Theoretical approaches addressing the issue have appeared in recent years,85 some of which also proposed how to assess the problem experimentally. It has been suggested that photon correlation spectroscopy, which relies on coincidence measurements of photons emitted by a system excited by incoherent, thermal light, can provide similar information as 2D spectroscopy.88 Similarly, Hong–Ou–Mandel (HOM) interferometry measuring coincidences of entangled photons generated in a nonlinear crystal89 may, if the studied system is placed into one arm of the HOM interferometer, provide information about the behavior of the light-system interaction in a single-photon regime corresponding to the conditions experienced under sunlight.90 Other promising approaches using measurements of entangled photons to simulate sunlight conditions have been recently proposed,91 indicating that the coming decade may witness the dawn of experimental approaches that will be able to follow the response of a light-harvesting systems to incoherent sunlight by using quantum light.

Today, we know that the pigment system of photosynthetic organisms consists of two parts, one that collects the sunlight, called antenna, and another one, the reaction center (RC), that converts the transient excited state energy of the antenna to a long-lived electrochemical potential that drives all energy demanding processes of the organism.79,92–94 Both antenna and RC are composed of pigment–protein complexes. An antenna, or light-harvesting complex (LHC), often consists of several polypeptides and tens to hundreds (or even thousands) of pigment molecules, while a RC generally is smaller with a few polypeptides and usually six pigment molecules. A functional antenna/RC assembly is often called a photosynthetic unit (PSU) and can consist of many LHCs per RC. The physical processes that achieve the light to electrochemical potential conversion is excitation energy transfer in the antenna and ET within the RC. Depending on factors such as involved pigments, distances, and the nature of interaction between pigment molecules, the timescale of this conversion ranges from femtoseconds to nanoseconds. The light-induced processes of photosynthesis are often studied in isolated LHC or RC preparations, but in order to characterize the energy flow through a functional antenna, or antenna-RC assembly, photosynthetic membrane preparations containing intact PSUs or whole organisms (i.e., algae, bacteria, and leaves) must be studied. The latter may pose a challenge in spectroscopic measurements because of a large number of spectrally overlapping pigments and strong light scattering that inevitably results in additional noise in the experiment.

Photosynthetic pigment–protein complexes, as well as membrane preparations have been studied using various methods of optical spectroscopy long before ultrafast measurements became possible. With the help of, e.g., fluorescence yield and polarized spectroscopy measurements, microscopic molecular level information was pursued (see Ref. 79 and references therein). Even before high-resolution crystal structures of RCs and light-harvesting complexes became available, simple geometrical models of pigment–protein complexes (see, e.g., Ref. 95) and lattice models79 of whole PSUs or photosynthetic membranes were developed to retrieve this information.

Time-resolved measurements with temporal resolution capable of resolving the most elementary photophysical and chemical processes (<100 fs) were an important step toward identifying individual energy and ET steps, as well as energy and charge flow through extended pigment systems, but not until ultrafast dynamics were reconciled with high-resolution structures could pathways of energy and charge transport be identified with certainty (see, e.g., Refs. 96 and 97).

In order to illustrate the principles of light harvesting by photosynthetic antennas and energy conversion by RCs and to show how ultrafast spectroscopy has contributed to the understanding of the photosynthetic processes, we have chosen to discuss results for four main groups of photosynthetic organisms, cyanobacteria and red algae, green bacteria, purple bacteria and higher plants. This includes results for a number of isolated light-harvesting complexes, the two types of RCs, as well as results for photosynthetic membranes and intact organisms. The ET processes in the RCs are best studied in isolated RCs, because of often overlapping spectral properties of antenna and RC pigments, and since antenna to RC energy transfer is often understood to be a rate limiting process.

Photosynthetic organisms thrive under widely varying light conditions, from bright sunlight to a very dim glow of black smokers several kilometers down in the ocean. Our choice of organisms will illustrate nature's strategies to optimize the effectiveness of a particular organism under the conditions defined by intensity and spectral properties of light in its environment. On the individual pigment–protein complex level, our choice of organisms translates into the following LHCs: phycobilisomes of cyanobacteria and red algae, chlorosomes, the Fenna–Matthews–Olson (FMO) complex and B808–866 complex of green bacteria, LH1 and LH2 of purple bacteria, and the antenna complexes associated with photosystem I (PSI) and photosystem II (PSII) of plants. In all organisms, RCs are of either type I or type II and we will discuss results for both. Before we proceed with discussions of energy and ET processes in the photosynthetic pigment machinery, we make a short detour for considerations of the experimental conditions required for obtaining relevant information on light-driven energy and electron dynamics in photosynthetic pigment–protein complexes.

Already in the early days of ultrafast spectroscopy, the primary light-driven processes of photosynthesis were targets of several studies. It started with three studies of the charge separation (CS) in RCs of purple bacteria6,7,98 and it was reported that the primary and secondary charge separation steps occur in less than 6 and 200 ps, values that by now have been verified over and over again after hundreds of more detailed and precise measurements. Studies of the energy transfer processes in photosynthetic antennas turned out to be more difficult. Fluorescence lifetimes of antenna complexes or photosynthetic membranes were reported to be excitation pulse intensity dependent,99–101 and it was soon realized that the reason for this can be found in the fact that two or more excitations in a pigment system containing many pigment molecules coupled through energy transfer will extinguish each other until only one remains. The process called exciton–exciton annihilation is mentioned in Sec. II C, and there are two variants of it—singlet–singlet and singlet–triplet annihilation. Thus, if several pigment molecules are excited in an antenna system with coupled chromophores two excitations (excitons) will eventually meet on the same molecule (or a coupled oligomer) and generate a high-lying excited state. Such a state generally has a very short lifetime, limited by radiationless decay. The whole process results in a fast and non-exponential decay of the excited state population. This decay is solely a consequence of the simultaneous multiple excitation of the pigment system and obviously does not reflect the intrinsic decay properties of the population, because, as mentioned above, under natural conditions multiple excitation is not feasible. With the early low-repetition-rate high-energy (many photons) ps pulses, it was impossible to avoid these non-linear effects and obtain kinetics free of distorting exciton–exciton annihilation. Theoretical methods were, however, developed to benefit from the exciton–exciton annihilation, and the size of the pigment system, i.e., number of connected chromophores, could be estimated from the shape of a curve describing the dependence of integrated fluorescence yield on excitation pulse intensity.102–104 Other parameters that can be estimated from the annihilation curve are the nearest neighbor energy transfer rate and energy trapping time by the RC. Thus, although these properties could not be obtained from direct kinetic measurements, they could be estimated from the annihilation behavior. Despite a model dependence built into the analysis that obviously has limited accuracy of obtainable information, many photosynthetic systems were studied, e.g., chloroplasts,99 photosynthetic bacteria,105 and phycobilisomes106,107 and useful information was extracted. Excellent reviews of the annihilation phenomenon, theoretical models and experimental studies are given in Refs. 79 and 108. Distorting exciton annihilation effects on energy transfer kinetics of large pigment systems can obviously be eliminated by using excitation pulses of sufficiently low energy, such that only one pigment molecule per domain is excited.

In this section we start with a short discussion of excited state properties of photosynthetic pigments: chlorophylls, phycobilins, and carotenoids and proceed to discuss specific results for the various photosynthetic systems outlined above. We follow the same order of events as found in photosynthesis—light harvesting first followed by the charge separation in RC.

Knowledge of excited state properties of isolated pigments which play a role in photosynthesis is the key prerequisite for understanding the light-driven processes in photosynthesis. There are three types of pigments involved in photosynthesis: (1) chlorophyllides that are present in both LHCs and RCs in all photosynthetic organisms; (2) carotenoids, which are also found in the majority of LHCs and RCs, but there exist LHCs, such as FMO, without carotenoids; and (3) phycobilins that occur exclusively in phycobilisomes of cyanobacteria, red algae, and glaucophytes, described in Sec. III B. The three groups of pigments also differ in their diversity in photosynthetic organisms. Only four phycobilins are found in cyanobacteria and red algae, while 13 different chlorophyllides (six chlorophylls and seven bacteriochlorophylls) plus a few pheophytins, which are bacterio(chlorophylls) missing Mg atom, have been identified so far in various photosynthetic systems.75 The pigment diversity is by far the largest among carotenoids. Even though only five carotenoid species are found in plants, more than hundred different carotenoids have been reported in various LHCs of photosynthetic micro-organisms.109 

1. Chlorophylls and phycobilins

Among the various photosynthetic pigments found in nature, the chlorophyllides are the most abundant. They include chlorophylls, such as chlorophyll a and b, (Chl a, b) and pheophytins found in photosynthetic systems of higher plants, as well as bacteriochlorophylls, such as bacteriochlorophyll a and c (BChl a, c) and bacteriopheophytins found in some species of photosynthetic bacteria. The functions of chlorophyllides include energy transfer and charge separation, and thus they serve as principle cofactors for the primary functions of photosynthesis. Currently, six Chls and seven BChls have been identified in various photosynthetic organisms (see Fig. 3 for the structure of Chl a). Already in 1881 Engelmann detected the action spectrum of photosynthesis in filamentous green algae, which closely resembled the absorption spectra of the Chl a and Chl b molecules. The latest discovered chlorophyll, Chl f, was reported in 2010, and was found in cyanobacteria from a stromatolite colony.110 The absorption spectrum of all Chls consists of two main spectral bands: the Soret band peaking in the 350–480 nm spectral range, and the lowest energy Qy band extending from 630 to 720 nm for Chls and 700–1010 nm for BChls.75 It is important to notice that the properties of the lowest energy Q band states (Qy and Qx) of chlorophyllides control the functions of these molecules. In the early sixties, M. Gouterman provided the explanation of the electronic structure of porphyrin type molecules.111 In the Gouterman model, two independent electronic transitions named Qx and Qy with perpendicular transition dipole moments were identified. Later, with the help of polarization spectroscopy studies, it was shown that the angle between the Q transitions is closer to 70°.112 In the absorption spectrum, transitions to these electronic states are accompanied by vibronic transitions involving coupled vibrational modes. Through the years, chlorophylls have been subject to numerous spectroscopic studies, and it thus comes as a surprise that the position of the higher-energy transition Qx is still debated. In a seminal paper in 2013 Reimers and co-workers showed that available steady-state spectroscopic data, including absorption, emission, fluorescence excitation, linear dichroism, and magnetic circular dichroism, of 32 chlorophyllides can be fit with a relatively simple vibronic coupling model, involving one vibrational mode and strong coupling between Qy and Qx.113 Thus, it was suggested that in all of these molecules, the Qy and Qx states (transitions) are inseparably vibronically mixed. This means that the direction of transition dipole moments of the mixed states is also something in between x and y, in other words x-polarized absorption spreads through the whole Q band. However, finding an unambiguous experimental proof for this conjecture turned out to be difficult.

In addition to the singlet states discussed above, (B)Chls have lower lying triplet states, which can be populated via an intersystem crossing pathway from the Qy state. The yield of this process was measured a long time ago and was concluded to be in the 50%–60% range for (B)Chl a and (B)Chl b (see Ref. 114) but recent data employing femtosecond transient absorption spectroscopy suggest lower yields around 30%.115 Triplet states do not directly participate in photosynthetic functions, but (B)Chl triplets can sensitize dioxygen producing highly reactive singlet oxygen, which can damage the photosynthetic apparatus. In vivo, both (B)Chl triplets and singlet oxygen are deactivated by carotenoid molecules (see Sec. III A 2).

A range of time-resolved spectroscopy studies have been performed to measure chlorophyll excited state lifetimes (see, for example, Ref. 116). Typical lifetimes of the singlet excited state (Qy) are in the range of a few nanoseconds, whereas triplet states typically decay with a lifetime from tens of microseconds to a millisecond. The Qy lifetime is readily measured by time-resolved fluorescence, and the first values were measured more than 60 years ago, providing lifetimes of 5.1 and 3.9 ns for Chl a and Chl b, respectively.117 Since then, a number of reports provided the Qy lifetimes of all (B)Chls ranging from ∼2.5 ns (BChl b) to ∼6.5 ns (Chl a, BChl c).116,118 Much less is known about the lifetime of the states giving rise to the Soret band. The Soret lifetimes of Chl a in a few different solvents were reported,119 yielding values of ∼150 fs. Comparable relaxation times were obtained after Soret excitation of Chl a (145 fs) and Chl b (160 fs) in ethanol solution.120 

The energy relaxation between Qx and Qy states was estimated to take place on a sub-100 fs timescale.72,119,121,122 Coherent vibrational dynamics, i.e., vibrational wavepackets in these molecules have been also investigated using the 2DES technique.72,123,124 Before that, the nature of the excited states and involvement of vibrations have been extensively studied using high-resolution spectroscopy techniques (see Refs. 113 and 125 and references within). The lack of symmetry between the absorption and emission spectra, as well as the difference between the vibrational spectra in ground and excited states of Chls suggested the presence of vibronic mixing and that Condon and Born–Oppenheimer approximations are not valid for these molecules. More recently this was also suggested in the combined theoretical and anisotropy 2DES study of Chl a in solution.122 In 2020, with the help of a polarization-controlled 2DES experiment (Sec. II B 3 c), which is sensitive only to the signals from either purely electronic coherences or coherences excited via vibronically mixed transitions, vibronic mixing between the Qx and Qy transitions in Chl c was unambiguously identified.72 It was also discovered that several modes are responsible for the vibronic mixing. It seems reasonable to assume that vibronic mixing is present in all chlorophyll-type molecules as suggested by Reimers et al.,113 but this will have to be confirmed in future experimental studies. Furthermore, the effect of the vibronic mixing in chlorophylls on modulating or optimizing photosynthetic functions remains to be explored.

Phycobilins, which occur exclusively in phycobilisomes, are linear tetrapyrroles derived from the bile pigment biliverdin. Three major bilins are found in phycobilisomes: phycocyanobilin (Fig. 3) absorbing in the 600–670 nm region, phycoerythrobilin (540–570 nm), and phycourobilin (490–510 nm).126 Isolated phycobilins in solution have photophysical properties very different from those reported for phycobilins bound in phycobilisomes.127 The binding site in phycobiliproteins locks the phycobilins in a specific conformation, preventing the conformational disorder that exists in solution. Phycobilins in solution have low fluorescence quantum yields (<0.1). They exist in several ground state conformers which exhibit rich photophysics resulting in short excited-state lifetimes ranging from 3 to 100 ps.127–129 In contrast, the lifetime of phycobilins bound to phycobilisomes are >1 ns, resulting in fluorescence quantum yields of ∼0.5.126 

2. Carotenoids

Carotenoids feature a complex electronic structure, and ultrafast spectroscopy has been proven to be crucial to disentangle their excited state properties. Carotenoids (see Fig. 3 for the structure of β-carotene) are derived from linear polyenes. The characteristic structural feature is the presence of methyl groups attached to the polyene backbone. The methyls cause bending of the carotenoid backbone that is not present in polyenes.130,131 In addition to the methyl groups, a number of carotenoids contain various terminal groups, leading to a large variability of carotenoid structures, currently counting to nearly 1000. Carotenoids are very good colorants due to their high molar absorption coefficients (ε ∼ 105 M−1 cm−1), but at the same time, they are essentially non-fluorescent. This apparent mystery was resolved in 1972 when experimental132 and theoretical133 study of the polyene octatetraene reported the existence of a low-lying state, which was forbidden for a one-photon transition from the ground state. These two papers became a landmark of carotenoid photophysics studies, which flourished especially after the appearance of ultrafast lasers enabling sub-ps time resolution.

a. The “three-state model.”

Following the discovery of the low-lying carotenoid dark state, the carotenoid excited-state properties were described using a three-state model consisting of the absorbing state (S2), the low-lying dark state (S1) and the ground state (S0) (Fig. 4). These states are, in the C2h symmetry point group, often used to approximate the symmetry properties of polyenes and carotenoids, denoted by Ag (S0 and S1) and Bu+ symmetry labels.134 The same parity of the S0 and S1 states (both Ag) has been traditionally used to rationalize the forbiddenness of the S0–S1 transition;134–136 however, recent reports show that the picture is far more complicated.137,138 This standard three-state model is often used to describe carotenoid excited states even today, though a number of experimental and theoretical studies carried out during the past two decades suggested that some additional states, which will be described below, are needed.

Within the framework of the three-state model, carotenoids absorb light via the S0–S2 transition characterized by a large oscillator strength in the 400–550 nm spectral region, resulting in the typical yellow-to-red color of carotenoids. The excited S2 state is depopulated via internal conversion to the dark S1 state, which then decays, again non-radiatively, to the ground state. The first direct measurement of the S1 lifetime was reported by Wasielewski and Kispert,139 yielding S1 lifetimes of 5.2, 8.4, and 25.4 ps for the carotenoids canthaxanthin, β-carotene, and 8′-apo-β-carotenal. Direct measurements of the S2 lifetime were not possible until the time resolution of ultrafast spectroscopy broke the 200 fs limit. Shreve et al.140 obtained a 200–250 fs S2 lifetime of β-carotene in 1991, which was at the limit of their time resolution. Later, Kandori et al.141 in 1994 used fluorescence upconversion with 100 fs time resolution to measure the decay of weak β-carotene S2 fluorescence and obtained an S2 lifetime of 200 fs. This ultrafast relaxation scheme explains the extremely low fluorescence quantum yields of carotenoids that, with a few exceptions, do not exceed 10−4.142,143

Already the first measurement of a carotenoid S1 lifetime139 demonstrated that excited-state properties depend on carotenoid structure. The key structural feature determining the spectroscopic properties is the conjugation length, N. In the simplest approach, N is determined by the number of conjugated C=C bonds, but a number of experiments showed that this is valid only for linear carotenoids. If the conjugation is extended to terminal rings or even includes other groups such as C = O, a notion of effective conjugation length, Neff, must be introduced. Thus, even though β-carotene (Fig. 3) has 11 conjugated C=C bonds, those located within terminal rings do not fully contribute to the conjugation, because the rings are twisted with respect to the plane of the linear conjugated backbone.144–146Neff may vary even for carotenoids having the same conjugated system as demonstrated for renierapurpurin and isorenieratene (with the S1 lifetimes of 6.5 and 13 ps, respectively) that differ only in positions of methyl groups at their terminal ring. Because of this, the terminal rings have different orientation with respect to the conjugated backbone, affecting Neff.147 

Ultrafast spectroscopy has been the key tool to determine lifetimes of carotenoid excited states and their relation to the carotenoid structure. The S1 lifetimes are readily measurable via decay of the excited state absorption from the S1 state. The S1–Sn transition, which for most carotenoids occurs in the 500–700 nm spectral region,136 has an oscillator strength comparable to that of the S0–S2 transition, and it is a fingerprint of the S1 state, with its shape and lifetime characteristic for each carotenoid. To this date, the S1 lifetimes of more than 50 carotenoids occurring in nature, whose effective conjugations cover the range Neff ∼ 8–14, have been measured.136 It is well established that the S1 lifetime follows the energy gap law: the S1 energy drops with increasing conjugation, which results in shortening of the S1 lifetime.148 The longest S1 lifetimes measured for natural carotenoids are in the 160–180 ps range for the carotenoid peridinin (Neff ∼ 8),149,150 but only in nonpolar solvent since peridinin belongs to the family of keto-carotenoids whose lifetimes depend on solvent polarity (see below). The carotenoid diketospirilloxanthin (Neff ∼ 14) with the S1 lifetime of 0.8 ps (Ref. 151) is on the other side of the S1 lifetime range.

The measurements of natural carotenoids have been complemented by data obtained on a number of synthetic carotenoids, extending the Neff range. At the long end, synthetic analogs of β-carotene with 15 and 19 conjugated C=C bonds (Neff approximately 14 and 18) have S1 lifetimes of 1.1 and 0.5 ps, respectively.152,153 A synthetic homolog of zeaxanthin with 23 conjugated C=C bonds is the longest carotenoid whose S1 lifetime has been measured so far. Formally, this corresponds to Neff ∼ 22, but for such a long conjugated system, the spectroscopic difference between N and Neff is negligible.154 The N =23 zeaxanthin has an S1 lifetime of 200 fs.155 At the short end of carotenoid conjugation, excited-state properties of β-carotene homologs with Neff ∼ 5.5 and 3.9 were reported, yielding S1 lifetimes of ∼300 ps and 2.7 ns, respectively;152,153 for synthetic peridinin with Neff ∼ 4.5 an S1 lifetime of 2.9 ns has been reported in n-hexane.156 

The carotenoid S1 state was also a principal target for time-resolved Raman spectroscopy. Carotenoids have strong Raman signals associated with C–C and C=C stretching modes, which in the ground electronic state have frequencies of about 1150 and 1520 cm−1, respectively. The frequency of the ground state C=C stretching mode depends on Neff and has been often used to determine Neff.145,146 The first time-resolved Raman measurements with picosecond time resolution, however, showed that in the S1 state the C=C stretching mode is significantly up-shifted, yielding values as high as 1780 cm−1.157 This upshift, which results from strong vibronic coupling between the S1 and S0 states,158 was later confirmed by the first time-resolved Raman spectroscopy experiments with sub-ps time resolution,159 and carotenoids have become popular testing molecules in the rapidly developing field of femtosecond Raman spectroscopy (Sec. II B 3 a).160,161 Subsequent measurements of time-resolved Raman spectra on various carotenoids confirmed the existence of the characteristic upshifted C=C stretching mode in the S1 state, whose dynamics corresponded to the S1 lifetimes. The upshifted C=C stretch was reported for peridinin at ∼1700 cm−1,162 fucoxanthin at ∼1750 cm−1,163 β-carotene at ∼1780 cm−1,57,164–166 zeaxanthin at ∼1775–1790 cm−1,166–168 canthaxanthin and astaxanthin at ∼1770 cm−1,166 and spirilloxanthin at ∼1740 cm−1.169 Since the reported values are listed in order of increasing Neff, it appears that, in contrast to the C=C stretching mode in the ground state, there is no direct correlation between Neff and C=C stretching frequency in the S1 state.

While the S1 lifetimes exhibit a clear correlation with the effective conjugation length, the relation between carotenoid structure and S2 lifetime is more complicated. The S2 lifetime drops below 100 fs for some carotenoids, requiring very high time resolution in order to characterize it. The first measured S2 lifetimes of β-carotene (200 fs)140,141 were overestimated, as experiments with better time resolution provided sub-200 fs values.170 A thorough fluorescence upconversion study of the β-carotene S2 lifetime in a wide range of solvents171 showed that the S2 lifetime also follows the energy gap law between the S1 and S2 states. The S2 lifetime decreased with increasing solvent polarizability, which also decreased the S2–S1 energy gap. The S2 lifetime varied from 180 fs in n-hexane to 120 fs in quinoline, because the S2–S1 energy gap decreases with increasing polarizability.171 However, this works only for the same carotenoid in solvents with varying polarizability. The dependence of the S2 lifetime on Neff exhibits a peculiar behavior as it decreases with increasing Neff in the 9–13 interval, while the S2–S1 energy gap is predicted to increase with increasing Neff.134 Thus, the longest S2 lifetime of 240 fs was reported for carotenoids with Neff ∼ 7, while it drops to 70 fs for Neff ∼ 13.153 However, shortening the carotenoid even further decreased the S2 lifetime again, yielding 160 fs for Neff ∼ 6.153 This anomalous N-dependence of the S2 lifetime is one of the indications suggesting that the three-state model is not sufficient to explain all the subtleties of carotenoid excited-state dynamics, and additional states may be required to describe the relaxation dynamics and pathways (see below). However, a fully quantum mechanical model denoted as vibrational energy relaxation approach (VERA) developed recently172 explains the anomalous N-dependence of the S2 lifetime without invoking any intermediate state(s). Instead, the relative displacement of minima of S2 and S1 potential energy surfaces is the key parameter determining the relaxation rate.173 

Vibrational cooling is the last dynamical process described within the three-state model. The S2–S1 internal conversion leaves molecules in a hot S1 state, which relaxes on the sub-picosecond timescale. The S1 vibrational cooling was first described in two papers in 2002.174,175 These papers reported red-shifted and broadened S1–Sn transitions at early delay times due to the population of higher vibrational states of the S1 state. Relaxation of this band shape to the final relaxed S1–Sn spectral profile is associated with vibrational cooling in the S1 state that for β-carotene was observed to occur in 300–500 fs, depending slightly on solvent. Later, routine use of broadband detection combined with global fitting methods identified vibrational relaxation in essentially all carotenoids. Studies of a series of carotenoids with varying Neff revealed a systematic acceleration of S1 vibrational cooling with increasing Neff, from nearly 1 ps in neurosporene (Neff = 9) to 160 fs in spirilloxanthin (Neff = 13).176 

Relaxation within the S2 vibrational manifold has not been reliably identified so far. Reports targeting this process are scarce and despite earlier attempts using excitation to the higher vibrational levels of the S2 state, this issue remains controversial. Based on fluorescence upconversion data, Akimoto et al. reported a 35–40 fs time constant,177 and comparable values were obtained by Kerr-gate fluorescence spectroscopy.178 Since a conical intersection has been invoked as one of the possible features of the mechanisms of S2 depopulation,179,180 it is likely that if the Franck–Condon region is situated close to the intersection point, a full vibrational relaxation of the S2 state does not occur at all. Such a scenario was also suggested in a few other studies.173,181,182 Detailed characterization of this process is nevertheless hindered by limits of time resolution, as well as by introduction of other ultrafast (sub-50 fs) relaxation channels to other dark state(s) proposed in a number of studies (see below).

Ultrafast spectroscopy was also important in determining the energy of the forbidden S0–S1 transition. Since the S1 state is not directly accessible from the ground state, its energy has been for a long time a matter of considerable debate. Along with steady-state methods relying on measurements of extremely weak S1 fluorescence,183 or resonance Raman profiles of the S0–S1 transition,184 a new approach based on measurement of the spectral profile of, the presumably allowed, S1–S2 transition within the S1 lifetime was developed in 1999.185 The S1–S2 transition occurs in the near-IR region spanning the 900–1800 nm range for most natural carotenoids and comparison of the S1–S2 and S0–S2 spectral profiles has been used to determine the S1 energy of a number carotenoids.185–187 

b. Other states—beyond the three-state model

Even though the three-state model reasonably describes the basic dynamics of carotenoid excited states, already the first calculations using more advanced methods reported in Ref. 134 suggested that additional states are likely present in the lowest-energy manifold of excited states of carotenoids or polyenes (Fig. 4). The calculations predicted two other “dark” states occurring within the S1–S2 energy gap for some carotenoids with conjugation length in the 9–13 range. The first is a state with Bu symmetry, which should be located below the S2 state for Neff ≥ 10; the second is another state with Ag symmetry, which is expected to drop below the S2 state only for Neff ≥ 13.134,188 However, these relations are valid only for the ground state geometry and more recent calculations suggest that the state ordering might change upon deviations from the ground state geometry leading to changes in bond length alternation.189–192 

While the third Ag symmetry state likely has little effect on excited-state dynamics as it may interfere with the S2–S1–S0 relaxation scheme only for very long conjugated systems, the search for the spectroscopic signatures of the Bu state has been an important part of research in carotenoid photophysics.193 First, the Bu state should be within the S2–S1 energy gap for most carotenoids occurring in natural light-harvesting systems, and possible effects on energy transfer efficiency could be expected. Second, the Bu state was supposed to “cure” the anomalous N-dependence of the S2 lifetime, which does not follow the energy gap law, though some recent studies showed that it is possible to explain it without invoking an intermediate state.173 

The first report claiming the assignment of a spectral feature to the Bu state appeared in 2002 and used a femtosecond spectrometer with sub-20 fs time resolution.194 For the carotenoids lycopene and β-carotene, a spectral feature peaking around 1000 nm with an extremely short lifetime of ∼10 fs was assigned to an intermediate state tentatively denoted as Sx, invoking a S2–Sx–S1–S0 relaxation scheme. Such an extremely short-lived intermediate state was subsequently proposed also in other carotenoids with Neff ≥ 10.190,195–198 In most reports, the Sx feature was tentatively assigned to the Bu state, though an alternative hypothesis involving a state with Ag+ symmetry dropping below the S2 state at the S2 geometry has been also proposed.190 A further explanation of the intermediate Sx state was proposed by Beck's group, which included changes in carotenoid configuration into the relaxation scheme.199 In such a scheme, the Sx state is assigned to the energy minimum of the S2 potential surface corresponding to a twisted configuration reached upon onset of torsional motions induced by excitation.200,201 Despite the number of studies mentioned above, the precise mechanism of the S2–S1 relaxation is still a matter of debate. One of the limiting factors is the time resolution of the experiments, as the processes involving the possible intermediate state occur on sub-30 fs timescale. Also, the presence of a multitude of experimental artifacts in the pulse overlap region complicates the matter. The data from 2DES experiments addressing this issue are also controversial, and both presence190,198 and absence202 of the Sx intermediate state have been reported by 2DES for carotenoids with comparable Neff. It should be mentioned here that coherent 2DES is highly prone to artifacts in the pulse overlap range, which are often ignored.203 

Another intensely debated state beyond the three-state model is commonly denoted as S*. In contrast to Bu, the S* is readily identified via its characteristic excited-state absorption, squeezed between the ground state bleaching and the main S1–Sn excited state absorption band.193 This spectral feature, known as the S* signal, was first reported in 1995 in very long (Neff. ≈ 18) derivatives of β-carotene.152 The S* signal exhibited slower decay than the S1 state and was assigned to the hot ground state. However, this assignment was later challenged in a report demonstrating that the S* signal of the carotenoid spirilloxanthin bound to a light-harvesting complex serves as a precursor for triplet formation and, therefore, must be a separate excited state.204 These early papers identified two possible origins of the S* signal, hot ground state and some excited state with similar properties, yet differing from the S1 state. Although extensive research during the past 20 years has shed some light on the possible origin of the S* state, a conclusive answer is still missing.

The S* lifetime and its relation to the S1 lifetime are important factors for understanding the overall carotenoid relaxation to the ground state. A number of studies provided evidence that for carotenoids with Neff ≥ 12, the S* lifetime is always longer than the S1 lifetime,152,155,204–206 while for shorter ones the S* signal decays with the same lifetime as the S1 state.181,207 This distinction provided a basis for kinetic modeling that showed that the S* signal could be successfully modeled by a combination of contributions from a hot ground state and the S1 state.207–209 The model of hot ground state was further elaborated by more sophisticated modeling using the vibrational redistribution approach VERA,172 showing that for carotenoids with Neff < 11 (such as, e.g., β-carotene) the dominating contribution to the S* signal comes from the S1 state, but with increasing conjugation length the hot ground state contribution prevails, because the S1 lifetime becomes shorter than vibrational relaxation in the ground state.210,211

While these reports seem to resolve the S* signal origin as due to a hot ground state, it must be noted that alternative models assigning the S* signal to a separate excited state can also reproduce the data. In these models, the S* state is usually associated with a distinct minimum on the S1 potential surface, corresponding to a “distorted” S1 state.176,205,212 Furthermore, while the hot ground state model is feasible for carotenoids in solution, it is hardly applicable for the S* signal reported for carotenoids bound to proteins. There, S* has been shown to serve as a precursor to triplet formation,204 as a minor energy transfer donor,213 and recently also as a quencher of Chl a excited states,214 or even as a state initiating photoconversion of the orange-carotenoid protein.215 Clearly, a hot ground state does not have sufficient energy to carry out these processes. Moreover, the S* signal in protein complexes containing carotenoids and (B)Chls may overlap with spectral features resulting from carotenoid electrochromic shifts resulting from excited (B)Chls nearby, further complicating the analysis of the S* signal origin.216,217

Finally, yet another state beyond the three-state model has been identified in keto-carotenoids, which contain a conjugated keto group in their structure. The keto-oxygen introduces asymmetry in the electron distribution along the conjugated chain, resulting in strong dependence of excited-state properties on solvent polarity. The polarity-dependent behavior was first reported for the carotenoid peridinin149 and subsequently detected in other keto-carotenoids. In polar solvents, a new excited-state absorption band in transient absorption spectra indicates the presence of an intramolecular charge transfer (ICT) state.149 Its presence in the excited-state manifold is usually associated with significant shortening of the carotenoid excited state lifetime.149,218 Later, another spectral signature of the ICT state, stimulated emission in the near-IR region, was discovered.150,219

The relation between the ICT and S1 states has been a matter of considerable debate, because these two states, clearly distinguished by their specific spectral bands, decay with the same lifetime.218,219 Therefore, a strong S1–ICT interaction resulting in a coupled S1/ICT state was assumed, with S1 and ICT corresponding to two minima on the same potential surface, separated by a low barrier.220 This picture has been only recently confirmed by pump-dump-probe spectroscopy, which allowed disturbing selectively the ICT part of the S1/ICT potential surface.163,221

Cyanobacteria and red algae can be found in almost every terrestrial and aquatic habitat. Phycobilisomes absorb in the range of 450 to 650 nm and are the main photosynthetic antenna of cyanobacteria, red algae and glaucophytes, attached to the outer, stromal, side of the photosynthetic membrane.126 They are composed of two similar phycobiliprotein units, α and β, to which phycobilin pigments are attached. The most common pigment–protein complexes are phycoerythrin, phycocyanin, and allophycocyanin, which aggregate into disk-shaped trimers or hexamers, forming the two major structural entities of the phycobilisome, the core and rods. The core, attached to the photosynthetic membrane, generally consists of three cylinders of stacked hexameric disks of allophycocyanin, to which a varying number of rods with a varying number of phycoerythrin and phycocyanin disks are attached.222–224 Phycobilisomes were thought to mainly deliver excitation energy to the PSII RC,126,223 but more recent work has shown that both PSI and PSII accept excitations from the phycobilisomes.225 This is illustrated by the phycobilisome structural model in Fig. 5, based on x-ray crystal structures of phycocyanin, allophycocyanin, and the PSI and PSII RCs of cyanobacteria.225,226

Non-time-resolved biochemistry and spectroscopy work showed that phycobilin pigments in the rods are organized in order of decreasing excited state energy from the outer tip toward the core, and fluorescence originates almost exclusively from allophycocyanin in the core.126,223,227 The energy gradient is created with phycoerythrin at the outer parts of the rods and phycocyanin further down the rods toward the attachment to the core. Further fine tuning of the energy gradient is achieved by pigment–protein interactions and by pigment-less linker proteins between the disks. This already suggested that efficient directional energy transfer occurs from the outer parts of the rods toward the core and then further to the RCs.

Early time-resolved fluorescence measurements on phycobilisomes from Porphyridium cruentum, having rods with both phycoerythrin and phycocyanin, seemed to verify this picture. At sufficiently low excitation pulse energy to avoid exciton annihilation, phycoerythrin fluorescence decayed with a 70 ps lifetime and the main allophycocyanin fluorescence was seen to have a rise time of 120 ps and decay of 4 ns.106,107 Somewhat similar results were observed in Ref. 228 for phycobilisomes from the same organism. Picosecond fluorescence measurements with low-energy dye laser excitation pulses revealed ∼60 and ∼40 ps energy transfer times from phycoerythrin and phycocyanin, respectively. The shorter energy transfer time from phycocyanin to allophycocyanin in the latter study is probably a result of the different mode of detection in the two studies—as rise time in Refs. 106 and 107 and decay time in Ref. 228. The overall energy transfer from the outer parts of the phycoerythrin rods to the terminal allophycocyanin emitter was concluded to be 70 ps, in better agreement with the allophycocyanin rise time observed in Ref. 107. A transient absorption anisotropy decay of 12 ps was assigned to phycoerythrin intra-heximer energy transfer.228 Still another time-resolved fluorescence study, this time on intact cells of the red algae P. cruentum, as well as the cyanobacterium Anacystis nidulans agreed with the above results,229 and nicely illustrated the sequential energy transfer through the phycobilisome rods, to the allophycocyanin core and finally to RCs. From the temporal evolution of the fluorescence spectrum, energy transfer was concluded to occur with a non-exponential decay law with 1/e decay times of 93, 132, 156 ps for the phycoerythrin-phycocyanin-allophycocyanin-Chl transfer steps of P. cruentum. In A. nidulans, the phycocyanin-allophycocyanin-Chl steps were seen to be somewhat slower.

Isolated phycobilisomes of another red algae, Rhodella violacea, exhibited a similar energy transfer behavior as observed for P. cruentum—non-exponential energy transfer with a dominating 34 ps component from phycoerythrin and 25 ps from phycocyanin.230 

Picosecond fluorescence and absorption studies of isolated phycobilisomes of the cyanobacteria Synechoccocus 6301 further adds to the energy transfer picture painted above. By detecting decay of phycocyanin fluorescence and rise of allophycocyanin fluorescence, it was concluded that overall energy transfer from phycocyanin to allophycocyanin occurs in 120 ps.231 These results were further corroborated by picosecond absorption measurements, verifying the overall rod to core transfer time of 80–120 ps;232 such measurements on the mutant AN 112 having rods of only one hexameric disk (instead of three) resulted in a shorter, 45–50 ps, phycocyanin decay time, further supporting the interpretation of rod-core energy transfer time. Energy transfer from the main pool to the final allophycocyanin emitter, transferring energy to the RC, was concluded to occur in 50 ps. Intra-disk energy transfer, both in the rods and the core, was concluded to be very fast, <10–15 ps.232,233 Similar results, <8 ps intradisk and 25 ps rod interdisk transfer, were reported by Glazer and co-workers in Ref. 234.

Much more recent measurements have been performed with higher time resolution and more sophisticated analysis. Thus, with the help of femtosecond transient absorption and picosecond fluorescence streak camera measurements and global analysis using a compartment model of the phycobilisome pigments, energy transfer was studied in isolated phycobilisomes of Synechocystis PCC 6803.235 It was found that intra-hexamer energy transfer in both phycocyanin and allophycocyanin occur within ∼2–5 ps, intra-rod equilibration 15 ps, overall rod to core transfer 50–70 ps, equilibration within an allophycocyanin cylinder 13–19 ps, and the slowest process is intercylinder equilibration in the core, 75–200 ps.

A recent whole cell time-resolved fluorescence study of Synechococcus WH 7803235 provides a detailed view of the overall energy transfer in the phycobilisome and trapping by the RCs. For Synechococcus, WH 7803 interdisk energy transfer between two different spectral forms of phycoerythrin was found to occur in 36 ps, from phycoerythrin to phycocyanin energy flows with a major fast time constant of 15 ps and a minor one of 90 ps. Energy transfer from phycocyanin to the allophycocyanin core is characterized by a 32 ps time constant and equilibration among the two allophycocyanin spectral forms occurs with a 25 ps time constant. The fastest step is a branched transfer from the low-energy allophycocyanin form (APC 680) to PSI and PSII with a combined time constant of 7 ps (11 ps to PSI and 20 ps to PSII). This study can be compared with that on P. cruentum performed 34 years earlier by Yamazaki and co-workers.229 The phycobilisomes of the two organisms are similar in that they both contain the three pigments phycoerythrin, phycocyanin, and allophycocyanin and we can see that the overall rod-to-core energy equilibration time is also similar, 100-ps timescale. However, the last energy transfer step, from the allophycocyanin core to chlorophyll pigments in the membrane, appears to be much faster in Synechococcus WH 7803, 7 ps vs 156 ps in Porphyridium.

What ultrafast spectroscopy taught us about cyanobacteria and red algae

Taken together, the measurements performed over 40 years, on both isolated phycobilisomes and intact cells of several different species firmly illustrate the function of phycobilisomes—light harvesting in the visible part of the spectrum, picosecond timescale directional energy transfer from high-energy pigments located at the peripheral edges of the phycobilisome toward the core attaching the phycobilisome to the membrane, and finally efficient energy transfer to chlorophyll pigments in the thylakoid membrane. The sequence is completed with an overall efficiency of >95%.236 It is also interesting to notice that early measurements at the outset of time-resolved studies of phycobilisome energy transfer could present a correct picture of the overall dynamics, and that more recent measurements with higher time resolution and more sophisticated analysis schemes have essentially confirmed the measurements performed 35–40 years ago, but also yielded a more detailed picture of the internal energy transfer dynamics of the phycobilisome. From the results discussed here it appears that the phycobilisome is a very efficient light-harvesting antenna, as has also been discussed in much more detail elsewhere.126,236 One might ask the question why evolution disposed of phycobilisomes in green algae and plants? In Ref. 236, it is speculated that the reasons might not have been related to efficiency of light harvesting, but to photosynthetic membrane structure and the separation of PSI and PSII to different regions in the thylakoid membrane of these more advanced organisms.

1. Structures and organization

Green bacteria are found mostly in hot springs, in sulfur-rich environments, often living at low light intensities. Green sulfur bacteria have been found in depths of up to 145 m in the Black Sea, and a species has been found living near a black smoker off the coast of Mexico at a depth of 2500 m. At this depth, the bacterium lives of the dim glow from the thermal vent since no sunlight can penetrate there. Chlorosomes (Fig. 6) are the main light-harvesting antenna of green sulfur bacteria (Chlorobi), green filamentous bacteria (Chloroflexi), and phototropic acidobacteria (Candidatus Chloracidobacterium). They contain BChl c, d, and e as the main light-harvesting pigments, which aggregates into large supramolecular complexes that may organize into different shapes, e.g., lamellae, tubes, spirals, determined by details in the substitution pattern of the BChl molecules. Unlike all other photosynthetic complexes, the organization and functional tuning of the pigments are controlled by long range pigment–pigment interactions, rather than pigment–protein interactions. Thus, within the BChl aggregates there are no or very little protein, but there are other molecules, carotenoids, and quinones with light-harvesting, redox control and protective functions; these molecules may also assist in the aggregation of the BChls. The BChl aggregates along with carotenoids and quinones are enclosed in an ellipsoidal sac of a lipid monolayer membrane, 100–200 nm long and 10–60 nm wide and thick. This membrane holds a range of proteins, often with poorly known (or even unknown) function. The most important protein is the CsmA protein, binding BChl a molecules and forming the baseplate on one side of the chlorosome. As we will discuss in more detail below, the baseplate is the conduit of energy flow from the internal chlorosome BChls to the next energy acceptor. In Chlorobi and Candidatus Chloracidobacterium thermophilum this is the Fenna–Matthews–Olson (FMO) pigment–protein complex, whereas in Chloroflexi it is the membrane bound B808–866 complex. FMO and B808–866 then finally transfer the energy to the RC pigments. See, for example, the following references for more information about chlorosome chemical composition and structure:237–244 

The FMO complex is the first chlorine-containing protein complex to have its three-dimensional structure resolved to high resolution and named after the scientists who first identified the complex, Olson,245,246 and performed the structural work, Fenna and Matthews.247,248 The protein is a homo-trimer holding seven BChl a molecules per monomer; a recently identified eighth BChl a is located at the interface between the monomers.249 Due to the relatively small number of pigment molecules, the FMO protein has become one of the best studied photosynthetic proteins and somewhat of a test ground for experimental and theoretical energy transfer studies.97,250

Green filamentous bacteria (Chloroflexi) lack the FMO protein that in green sulfur bacteria interface the chlorosome to the photosynthetic membrane and RC complex. Instead, there is an integral membrane protein resembling a hybrid between the LH1 and LH2 protein complexes of purple bacteria. Electron microscopy studies of the green filamentous bacterium Roseiflexus castenholzii, which lacks chlorosomes, showed that it consists of ∼15 α/β subunits surrounding the RC and holding three BChl a and two carotenoid molecules per subunit.251,252 In R. castenholzii the BChl a molecules give rise to two absorption bands, one at 800 nm (B800-like of LH2) and another at 880 nm (LH1-like); in Chloroflexus aurantiacus, this protein complex has a bit different absorption spectrum, B808–866.242 

2. Energy transfer in chlorosomes

The aggregation of BChl molecules in chlorosomes and the variation of aggregate size and shape, discussed above, could suggest ultrafast intra-chlorosome energy transfer (exciton relaxation) as well as considerable variation in the energy transfer dynamics depending on bacterial species of origin of the chlorosome. The large number of studies of chlorosome energy transfer by many groups seem to verify this expectation. Rather than discussing all individual studies one by one we will identify common results and general trends, and where there are significant differences we try to understand the reasons. Experimental methods of choice to achieve the expectedly necessary femtosecond temporal resolution is in most studies transient absorption or two-dimensional electronic spectroscopy. All studies agree that the energy relaxation occurs over a wide time range. The fastest processes on the few-hundred fs timescale are assigned to relaxation from high energy exciton states to lower-lying states.243,253–255 In addition, all studies agree that there are slower, 10–20 ps, processes, but other, both faster (∼1 ps) and slower (∼30 ps), dynamics are reported. These processes are generally assigned to relaxation among low-lying exciton states,77,253,256 or alternatively expressed, between exciton levels located on different BChl layers of the chlorosome lamellae. The spread in lifetimes for the slow components is perhaps not surprising considering that studies were performed on chlorosomes from both Chlorobi and Chloroflexi containing both BChl c and BChl e, with the possible variation in BChl c/e aggregate structure. A 2D electronic spectroscopy254 work showed that incoherent exciton diffusion within and between coherent domains occurs on a sub-100 fs timescale. Most of the distribution of exciton energies are sampled within 200 fs, but minor evolution of the 2DES spectrum on a slower timescale shows that there are other slower energy transfer processes, as suggested in earlier studies.

Theoretical simulations of exciton dynamics point in the same direction as the experiments regarding the ultrafast part of the relaxation processes. Thus, exciton delocalization was concluded to be complete in 100–200 fs (Refs. 255 and 257) and energy relaxation in the exciton manifold in ∼50 fs. For excitonically coupled pigment molecules, the exciton delocalization size is a quantity reporting on properties of the aggregate (e.g., pigment–pigment coupling, energy disorder, dynamic disorder, etc.).258–260 The photoinduced bleaching in the BChl c Qy absorption band of chlorosomes from C. aurantiacus was observed to be 7–8 times greater than that of the BChl a band of the chlorosome. This was interpreted as proof of exciton delocalization over many BChl c molecules. These results were further analyzed with exciton theory using an aggregation model of the BChl c molecules261 and found to correspond to an aggregate size of 24 BChl c molecules arranged as a tubular aggregate of six linear chains of four BChl molecules with an exciton delocalization size (defined as inverse participation ratio of the density matrix) of 7.4 at room temperature.260,262

The BChl a baseplate of the chlorosome is the gateway out for the energy from the BChl c (d or e) interior of the chlorosome. The transfer can be timed by measuring the appearance of BChl a excitations, or the overall decay of BChl c excited states. Since the baseplate is an integral part of the chlorosome, this time should be obtainable from measurements on isolated chlorosomes, but it is interesting to compare it with the corresponding measurements on membranes or intact cells to see if the interactions with the photosynthetic membrane and proteins attached to it (e.g., FMO) somehow influence the energy transfer. BChl c(d, e) to baseplate BChl a energy transfer has been studied in both Chlorobi and Chloroflexi isolated chlorosomes. Despite a significant spread of measured lifetimes in different studies, it seems that the BChl c(d, e) to baseplate energy transfer is faster in Chloroflexi, 8–40 ps (Refs. 253, 263, and 264) than in Chlorobi, ∼70–140 ps.77,253,256,263 In some studies, for chlorosomes of both taxa, a minor 2–10 ps component in the BChl c(d, e) to baseplate energy transfer was also detected.77,256,264 The faster energy transfer in Chloroflexi could perhaps be related to the bigger chlorosomes of the Chlorobi taxa.244 Alternatively, BChl that is not BChl c in the chlorosome could also lead to a different spectral overlap and slower energy transfer. Three different studies on whole cells of C. aurantiacus all report 15–16 ps for the BChl c to baseplate energy transfer.263,265,266 Thus, there appears not to be a significant difference in the transfer time measured in isolated chlorosomes and whole cells.

There are relatively fewer direct studies of intra-baseplate energy transfer studies, but one report256 assigns a transient absorption anisotropy decay at 807 nm in chlorosomes of Chlorobaculum tepidum of ∼1 ps to energy transfer between BChl a molecules in the baseplate. A 2DES study on chlorosomes of the same bacterium at 77 K could resolve relaxation processes through four exciton states, originating from four BChl a molecules that likely interact strongly, on the timescale of <1 ps to 20 ps. The faster ∼1 ps relaxation within the higher-lying states, and the slow ∼20 ps among the two lowest states.267 Thus, it appears that energy relaxation and equilibration within the BChl a baseplate is fast, particularly at room temperature. This, and the fact that BChl c(d,e) to BChl a transfer is much slower in all chlorosomes makes it difficult to observe this process except with direct BChl a excitation.267 It is worth noting that the electronic structure found in the baseplate is similar to that found in the FMO complex and the RC core antenna. This led authors to propose the feasibility of “lateral” energy transfer through the system, where excitation does not necessarily relax to the lowest state in the baseplate or FMO, before it is transferred to FMO or RC, respectively.267 

As mentioned above, chlorosomes contain carotenoids having light-harvesting and photoprotection functions.11,268 A few studies have revealed ultrafast, ∼100 fs energy transfer from the carotenoid S2 state to BChl c with 50%–80% efficiency.268,269 Carotenoid to BChl c energy transfer was also studied in synthetic BChl c aggregates270,271 and similar to the work of Psencik et al.269 it was found to occur from the S2 state, but somewhat slower, ∼500 fs, and less efficiently 30%–40%. The carotenoid molecules were also found to influence the aggregation of the BChl c molecules in the synthetic aggregates.271 

3. Energy transfer in the Fenna–Matthews–Olson and B808–866 complexes

The group of Struve (and later together with Savikhin) were some of the first to study FMO energy transfer dynamics. In two early reports, using few-ps dye laser pulses in one-color transient absorption measurements,272,273 anisotropy decays of 2.3–4.7 ps were measured and interpreted as inter-monomer energy transfer in the trimeric FMO protein. The reason for this interpretation was that intra-monomer energy transfer (or exciton relaxation) between the strongly interacting BChl c molecules was considered too fast to be resolved with the picosecond resolution of the experiment. With the advent of much shorter Ti:Sapphire laser pulses, these experiments were repeated with better temporal resolution,274,275 and faster relaxation processes could be resolved. Thus, 100–900 fs lifetime components in isotropic and anisotropic transient absorption measurements were interpreted as energy equilibration between higher lying exciton states, and a slower anisotropy decay phase of 1.4–2.0 ps was tentatively assigned to equilibration among the lowest energy pigments.274,275 Further broadband measurements of transient absorption spectra with several different excitation wavelengths within the FMO absorption band at 17 K resulted in a level to level relaxation model.96,276 Decay from upper to intermediate exciton states was found to occur in ∼30–100 fs, while the further decay to the lowest exciton states was concluded to be considerably slower, ∼0.6–2.5 ps. At room temperature, these processes, in particular the decay to the lowest states, would presumably be faster. There is a good agreement in reported time constants from all studies, but the early assignment of the few-ps processes to inter-monomer energy transfer272,273 now appears to be intra-monomer decay between the lowest exciton levels. However, we need to keep in mind that the early measurements were performed at room temperature, whereas the later ones were at 17 K, so the agreement may still be a coincidence. As a concluding remark, in Ref. 96 it was mentioned that “a major impediment to molecular level interpretations of such experiments is the absence of a reliable theory for protein site effects on pigment properties…” and “New strategies for determining the antenna electronic structure (e.g., by independent nonlinear optical techniques) would be valuable.”276 

Nineteen years, later this was achieved with the help of 2D electronic spectroscopy (2DES). Thus, with 2DES and full polarization control at 77 K an accurate description of electronic structure, with state to state correlation, and energy transfer dynamics was obtained.97 The recently discovered eighth BChl a molecule could in addition be spectroscopically identified. The energy transfer scheme obtained from the 2DES measurements is similar to that generated previously by more traditional transient absorption measurements,276,277 but the level-to-level scheme is more detailed. Thus, the energy transfer dynamics corresponds to a picture of energy flow between the eight BChl c molecules of the FMO monomer.

The energy transfer dynamics discussed above were obtained for FMO proteins isolated from two different green sulfur bacteria, C. tepidum and Prosthecochloris aestuarii, so it cannot be excluded that some differences in the reported dynamics originates from this difference in origin.

The ideas that superpositions between the excitons may play a role in photosynthetic energy transfer dates back to the 1960s.278 The observation of coherent oscillations in transient absorption anisotropy measurements of the FMO complex of C. tepidum at 19 K was a first hint that such effects may be present.279 The oscillations had a period of 220 fs, and a dephasing time of 140–180 fs and were almost absent in the magic angle signal; they were of much higher amplitude when the excitation pulse spectrum covered both the 815 and 825 nm bands of the absorption spectrum, compared to when only one of the bands was excited. These features led the authors to conclude that the oscillations were a result of quantum beats between two exciton states, rather than due to vibrational coherence. However, the authors did not discuss possible implications of these findings for energy transfer in the FMO protein. Later, in 2007, oscillations were claimed to persist for up to a picosecond in 2DES measurements on the FMO protein at 77 K,83 and interpreted as electronic coherences. These results were taken as the signature of wavelike characteristics of energy transfer, explaining its high efficiency. This has been shown to be incorrect in later studies. The topic is discussed in somewhat more detail in Sec. III H.

Energy transfer within the B808–866 protein in chlorosome-membrane complexes from C. aurantiacus were studied in two-color pump-probe experiments at room temperature.280 The B808–B866 energy transfer time was found to be 2 ps, somewhat slower than B800–850 transfer in LH2 (0.7 ps at room temperature, see Sec. III D 2 on purple bacteria and Ref. 281). A somewhat longer transfer time, 5 ps, was obtained from time-resolved fluorescence measurements.282 Transient absorption measurements on the closely related B800–880 complex of R. castenholzii283 produced the same order-of-magnitude lifetime of 2.3 ps. For B808–866, no intra-B808 energy transfer was found, in contrast to LH2, where intra-B00 transfer was found to proceed on the ∼0.5 ps timescale.281 Intra-B866 dynamics was found to be characterized by a time constant of 350–550 fs, again slower than in LH2 and LH1 of purple bacteria.281 Carotenoid to BChl a energy transfer was studied in the B800–880 complex of R. castenholzii; for the two dominating carotenoids (methoxy/hydroxyl-γ-carotene and methoxy-keto-myxocoxanthin) S2 to BChl transfer was found to occur with 38 (17)% efficiency and transfer times of 180 (430) fs, whereas S1 to BChl transfer proceeds with an efficiency of 19 (17)% and transfer time 10.4 (8.3) ps. Both carotenoid-to-BChl a energy transfer channels in this complex are thus less efficient and slower than in LH2 complexes (see below).

4. Energy transfer through the complete PSU

From the accounts above, we now have some insight into the energy transfer dynamics within individual light-harvesting complexes of green bacteria, but we would also like to know how these complexes are coupled together and the resulting dynamics of energy flow between them and into the RC. For this, studies of energy transfer of intact organisms or at least photosynthetic membranes containing an intact functioning PSU are required. This implies measurement of decay and rise of excited state populations of all the LH complexes (and RC) of the PSU. An efficient energy transfer chain necessarily implies spectral overlap, i.e., spectral congestion and, from an experimental point of view, difficulties to achieve selective excitation and probing. In a PSU, there is in addition often spectral overlap between antenna and RC pigments, further adding to the complexity. As already mentioned above, energy transfer to the RC is often rate limiting, making it difficult to access the fastest CT processes in RCs from measurements on intact organisms. We will discuss this point further in the sections on RC processes.

The difficulties to resolve the energy transfer steps between LH complexes appear to have been rather difficult in intact green sulfur bacteria. Apart from a very recent 2DES study on C. tepidum,284 no detailed reports are available. In this study, in addition to intra-complex energy transfer, chlorosome to FMO energy transfer was reported to occur with a time constant of 70 ps, and FMO to membrane pigment transfer with a dominating lifetime of 17 ps. The efficiency of the FMO to membrane energy transfer step was reported to be ∼75%,284 considerably higher than the 40%–50% reported from studies of a RC-FMO complex or membrane preparations (see, e.g., Refs. 242, 285, and 286). In Ref. 285, 66% of the FMOs of the RC-FMO preparation were inactive in energy transfer, and the 34% of the FMOs that did transfer energy to the RC did so with 76% efficiency. Observed differences could suggest that the isolation procedures of the complex and membrane preparations adversely affected the coupling between FMO and the photosynthetic membrane. Additionally, measurements at physiological temperatures through the intact PSU are required to provide the final answer regarding the energy transfer efficiency.

The direct attachment of chlorosomes to the photosynthetic membrane in green filamentous bacteria, without an interfacing FMO complex, makes energy flow through the antenna system less complex. Energy leaves the chlorosome through the BChl a baseplate and enters the membrane through the RC core complex (B808–866 in C. aurantiacus), with direct access to the RC.251,252 This means that knowing the ultrafast internal energy dynamics of chlorosomes and the RC core complex, measurements of decay of baseplate excitations, or rise time of core complex excited states reflect the overall energy transfer to RC-coupled pigments. This was done already 30 years ago in three different time-resolved fluorescence measurements,263,265,266 all showing that the overall energy transfer time is ∼40 ps. The further trapping of the energy by the RC in C. aurantiacus was reported to be 43,263 or 70–90 ps.265 In Ref. 266, the RC trapping could equally well be characterized by a single exponential decay of B866 fluorescence with 140 ps lifetime, or a double exponential with the lifetimes 70 and 200 ps of approximately the same amplitude. These two lifetimes agree perfectly with the lifetimes reported in Ref. 265 for open (70–90 ps) and closed (secondary electron acceptor QA reduced) RCs (180–200 ps), suggesting that the sample in Ref. 266 had a mixture of open and closed RCs. These three studies together show that the overall energy transfer from chlorosomes to the RC core antenna in green filamentous bacteria proceeds with a time constant of ∼50 ps and further trapping by the photochemically active RC takes about the same time. It is important to realize that the trapping time does not represent direct energy transfer from B866 to the primary electron donor in the RC, P, but the effective time it takes for the RC to secure the energy through electron transfer to the primary pheophytin electron acceptor. From modeling of the measured kinetics, it was concluded that the excitation from the antenna visits the special pair (P) in the RC five to ten times before primary charge separation occurs, because back energy transfer from P to B866 is about ten times faster than forward energy transfer.265 This type of energy dynamics is called trap-limited, in contrast to diffusion-limited where the overall energy diffusion to the RC is the rate limiting process. We will see that different types of organisms represent either of these two cases, depending on the organization of the antenna and antenna-RC interactions.

What ultrafast spectroscopy taught us about photosynthetic green bacteria

We have discussed two types of green bacteria—Chloroflexi and Chlorobi. The PSU of Chlorobi consists of a light-harvesting apparatus composed of chlorosomes with an integral baseplate connecting it to the FMO complex, which channels the energy collected by the more peripheral parts of the antenna into the RC-core antenna complex. In Chloroflexi bacteria, a membrane-bound B800–866 (or B800–880), reminiscent of purple bacterial LH2, replaces the FMO complex. Apart from that, the organization of the Chlorobi and Chloroflexi PSUs is similar. The overwhelming number of BChl pigment molecules in chlorosomes, as compared to the other antenna proteins and RC, combined with partially overlapping absorption spectra has made studies of energy transfer dynamics in intact green bacteria particularly challenging. Thus, most of the published results are for individual pigment–protein complexes. Nevertheless, the following picture emerges from the available results:

  • Densely packed and strongly interacting BChls in chlorosomes result in excitons delocalized over several molecules (the exact number is still debated), similar to what has been found for LH2 and LH1 of purple bacteria (see below).

  • Within a chlorosome, relaxation from high-energy exciton states to lower-lying states within a BChl layer of a lamella occurs on the few-hundred fs timescale, while relaxation between low-lying exciton states on different BChl layers is slower, 1–10 ps.

  • Energy absorbed by the BChls within the chlorosome ends up in the baseplate of the chlorosome within ∼15–100 ps, the longer times for the bigger chlorosomes of Chlorobi bacteria. Energy relaxation and equilibration within the baseplate proceeds on the 20 ps timescale.

  • Thanks to the relatively few (eight) BChl a molecules of the FMO complex in Chlorobi bacteria an accurate description of electronic structure and energy transfer dynamics with site-state correlation has been obtained. Energy equilibration between higher lying exciton states proceeds on the ∼50 fs timescale, and relaxation to and equilibration among low-lying exciton states takes 1–2 ps. The more than 10-year long debate on long-lived electronic coherence in FMO and its role for energy transfer efficiency appears to have been settled with the conclusion that the coherences are of vibronic origin, and thus no different than those observed in many other pigment systems with ultrashort pulse excitation (for details, see Sec. III H on coherences in photosynthetic systems, below).

  • In Chloroflexi bacteria, lacking FMO, chlorosomes are interfaced to the RC-core antenna complex in the membrane by the B800–866 (or B800–880) complex. Energy transfer within this complex is similar to that in LH2 of purple bacteria—∼2 ps energy transfer from B800 to B866/B880 and few-100 fs dynamics within B866/B880.

  • The difficulties in resolving the energy flow through the complete PSU of a green bacterium were mitigated with the help of 2DES experiments on intact C. tepidum. In addition to the intra-complex processes, the missing information on inter-complex energy transfer was achieved—chlorosome to FMO 70 ps; FMO to membrane pigments 17 ps at 77 K.

  • From all this, the overall energy transfer time from the peripheral antenna (chlorosomes) to RC core antenna in the membrane appears similar to the corresponding overall transfer time in purple bacteria (∼60 ps), discussed below, but the overall efficiency has been reported to be lower. The reason may partially be due to the very different organization of the PSU. Alternatively, more accurate experiments at physiological conditions might end up showing more efficient energy transfer.

1. Structural aspects

Photosynthetic purple non-sulfur bacteria are distributed widely in natural habitats, particularly in those with large amounts of soluble organic matter, such as swamps, wastewater ponds, coastal lagoons, and waste lagoons. Purple sulfur bacteria, on the other hand, are generally found in illuminated anoxic zones of lakes and other aquatic habitats where hydrogen sulfide accumulates and also in “sulfur springs” where geochemically or biologically produced hydrogen sulfide can trigger the formation of blooms of purple sulfur bacteria. Anoxic conditions are required for photosynthesis; these bacteria cannot thrive in oxygenated environments.

The photosynthetic unit of purple non-sulfur bacteria (in the following, just purple bacteria) consists of two different light-harvesting complexes, a peripheral LH2 antenna and a core LH1 antenna coupled to the RC. The high-resolution structures of two different LH2 complexes have been determined—LH2 of Rhodoblastus acidophilus (formerly Rhodopseudomonas acidophila) was solved in 1995 (Ref. 287) (Fig. 7) and that of Phaeospirillum molischianum (formerly Rhodospirillum molischianum) a year later.288 Both are a circular arrangement of transmembrane pairs of single helix α/β polypeptides, nine for R. acidophilus and eight for P. molischianum. Each α/β dimer holds three BChl a molecules and one carotenoid, thus in total 27 BChls and nine carotenoids for R. acidophilus (24 and 8, respectively, for P. molischianum). For R. acidophilus (P. molischianum) nine (eight) BChl a molecules are organized in a ring, at a Mg–Mg distance of 21 Å (very similar for P. molischianum), with the BChl macrocycles almost parallel to the photosynthetic membrane. This ring of nine (eight) BChl molecules is associated with the 800 nm absorption band and therefore termed B800. The relatively long distance between the BChl molecules in the B800 ring leads to weak BChl–BChl interaction, estimated to ∼25 cm−1 (see, e.g., Refs. 281 and 289 and references therein). Eighteen (R. acidophilus), or 16 BChls (P. molischianum) collectively absorbing at ∼850 nm are organized in another circle (B850), with molecular planes perpendicular to the membrane. The Mg–Mg distances are much shorter in B850, ∼9 Å, leading to much stronger BChl–BChl interaction estimated to be on the order of 300 cm−1 (see, e.g., Ref. 281 and references therein). The carotenoid molecules run between the α and β polypeptides, perpendicular to the plane of the membrane.

LH1 has a similar arrangement of α/β polypeptide transmembrane helices as LH2, with the RC encircled. Structures of LH1-RC complexes of several purple bacteria species have been determined, exhibiting a variation in size and shape (Fig. 8)—circular290 and elliptical291 encapsulating one RC, and S-shaped embracing two RCs.292 The elliptical LH1-RC complex of Rhodopseudomonas palustris291 has an opening in the sequence of 15 α/β polypeptide pairs, and the S-shaped LH1-RC complex of Rhodobacter sphaeroides292 has similar gaps at the ends of the “S.” These gaps in the LH1 continuity have been shown to be associated with a pigment-free protein, helix W,291 or pufX292 and to be involved in the trafficking of quinones in and out from the RC. The LH1-RC complexes have one ring of BChl a molecules, perpendicular to the membrane plane, i.e., similar to B850 of LH2. The pairwise BChl Mg–Mg distances are similar to those in B850 leading to an estimated interaction of ∼500 cm−1 (see, e.g., Ref. 281 and references therein). LH1 of several purple bacteria has a main absorption band at 875 nm (B875) related to the BChl a molecules. The LH1-RC complexes bind one carotenoid molecule per α/β dimer, similar to LH2. Structures of the three known types of LH1-RC complexes are shown in Fig. 8.

2. Energy transfer in light-harvesting complexes

During a period of approximately fifteen years from the mid-1980s to the end of the 1990s, with a peak during the years following the publishing of the LH2 structure,287,288 intense research by several groups contributed to a comprehensive and detailed picture of the energy flow through the antenna network of purple bacteria. Already before the high-resolution LH2 structures became available, a significant amount of data on excitation dynamics had been accumulated. When these data were combined with the structural information a dynamics-structure-function picture of the light-harvesting processes could rapidly be developed and further refined in the following years.281 Much more recent work and application of new experimental methods have added further important pieces of information to the description. Below, we summarize how the picture of purple bacterial light harvesting developed.

a. LH2: B800 to B850 energy transfer

We start by considering the energy transfer process in LH2 that contributes to the downhill energy flow toward the RC, B800–B850. This is one of the most studied and best characterized processes in photosynthetic light harvesting. Already, early picosecond absorption293 and fluorescence294 measurements on Rb. sphaeroides chromatophores at room temperature indicated that this process occurs with a time constant of ∼1 ps. Several later measurements with shorter sub-ps and fs pulses provided a more precise value of the time constant of this transfer step—0.7 ps at room temperature.295–302 These measurements were in addition performed on LH2 complexes of different purple bacteria species, showing that this energy transfer step is species independent (within experimental error of the measurements). In order to provide insight into the mechanism of B800–B850 energy transfer, a series of LH2 mutants with a successively blue-shifted B800 absorption band were studied. It was found that the B800–B850 energy transfer time gradually increases with increasing energy separation between the B800 and B850 bands, in qualitative (but not quantitative) agreement with Förster spectral overlap calculations.303,304 The temperature dependence of B800–B850 energy transfer was also examined and the transfer time was found to increase to 1.2 ps at 77 K (Refs. 300, 301, and 305) and 1.5 ps at 4 K.300 This is the expected trend for Förster energy transfer, due to a decreased spectral overlap at lower temperature. However, the measured transfer times were found to be considerably shorter than the calculated Förster transfer times, based on a quantitative spectral overlap calculation, and the temperature dependence was not reproduced by the calculations.300 To resolve this issue, it was proposed that spectral overlap between B800 and the upper exciton component of B850 provides an additional pathway of energy transfer.300,306,307

b. Energy transfer within the B800 ring

From the LH2 structure,287,288 distance between the BChl molecules of the B800 ring, and an estimate of nearest neighbor interaction between B800 molecules (∼25 cm−1),281 it can be concluded that B800 excited states are localized, but the interaction is still sufficiently strong for fast energy transfer. The first hint that it actually occurs comes from the observation that B800 fluorescence at 4 K is depolarized.95 In a time-resolved measurement, energy transfer within the B800 ring can be detected in two different ways, as a decay of time-resolved anisotropy as the excitation moves around the ring, or as an excitation wavelength dependent change of isotropic absorption or fluorescence intensity, if the absorption band is inhomogeneously broadened. In the latter case, excitation in the blue wing of the absorption spectrum would lead to a decay at short wavelengths and a corresponding rise of signal at long wavelengths. Both these features have been observed for B800. The first time-resolved absorption anisotropy study of B800 of two different LH2s of Rb. sphaeroides and R. palustris at room temperature revealed an ∼1 ps anisotropy decay,296 a similar anisotropy decay time was found for B800 of R. acidophilus.301 At 77 K, a shorter, ∼300 fs, depolarization time was observed,301,308 and master equation simulations describing the energy transfer as incoherent hopping in the ring of B800s, showed that the measured B800 dynamics could be described as energy transfer with a characteristic average nearest-neighbor pairwise transfer time of 0.35 ps. Several isotropic transient absorption measurements at 77 K revealed an ∼500 fs relaxation time interpreted as energy transfer from blue to red B800s.301,305,306,308,309 Considering all measured dynamics of the B800 band, it was concluded that “energy transfer within the B800 ring can be understood on the basis of the Förster equation for energy transfer in the weak coupling limit,” and “a neighbor-to-neighbor transfer time of 0.5–1 ps would be more than sufficient to account for the experimental observations.”281 This includes the longer transfer time at room temperature,296 as well as a factor of two longer B800-B800 transfer time in LH2 of P. molischianum.310 

Purple sulfur bacteria also contain LH1 and LH2 complexes, but much less is known about their structure, spectroscopy, and light-harvesting processes. Allochromatium vinosum (formerly Chromatium vinosum) is an interesting, somewhat peculiar species. Instead of the single B800 absorption band of purple non-sulfur bacteria, discussed so far, LH2 of the purple sulfur bacterium Alc. vinosum exhibits a split B800 absorption band.311,312 Different explanations to this have been offered; one assumes that there are two spectrally different LH2 complexes, impossible to separate with biochemical methods. Another explanation suggests that there are two different B800 BChl sites within the same LH2 complex, due to different pigment binding pockets. A third hypothesis is that the B800 BChls are organized in excitonically coupled dimers, as a consequence of reduced inter-chromophore distance, which results in a splitting of the absorption band.312 Non-conclusive and mutually conflicting results for these hypotheses have been presented.312,313 With the help of 2DES, this unsatisfactory state of affairs was resolved.314 The detection of a cross-peak between the two diagonal B800 peaks conclusively showed that there is excitonic coupling between the BChl molecules of the two B800 sub-bands and proved that the hypothesis of two spectrally different LH2 complexes does not hold. Nevertheless, it was concluded that this finding does not exclude that the BChl molecules of the two B800 bands could have different site energies.314 

c. Energy transfer within the B850 and LH1 rings

From the short BChl–BChl distances, ∼9 Å, in the B850 ring, it is immediately clear that the interaction between BChls is strong—with some variation of the precise value, it has been estimated to be on the order of 300 cm−1, and the inhomogeneous broadening of the same order of magnitude.281 This doubtlessly leads to excitonic delocalization of the excited states. The degree of exciton delocalization was a controversial issue during a few years following the publication of the LH2 structure.

There are several methods that can be used to access the size of the exciton. The magnitude of photobleaching (ground state to one-exciton transition), or induced absorption (one-exciton to two-exciton transition) of the antenna complex, as compared to the monomer pigment, is an apparently straightforward measure that has been used to conclude that the exciton is delocalized over a large part of the B850 and LH1 rings, or most likely the whole ring.315,316 The same photobleaching method, but with the B820 BChl dimer as reference was used to avoid sources of experimental uncertainties, and an exciton coherence length of 5 BChls was obtained.317 The transient absorption spectrum and the energy difference between the peaks of ground state bleaching and induced absorption is another measure318 that was used to estimate the size of the B850 exciton to 4 ± 2 BChls at 2 ps after excitation.319 This was confirmed by more sophisticated exciton calculations including diagonal energy disorder and fitting to experimental transient absorption spectra of B850 in the temperature range 4–296 K. At 1.5 ps after excitation, a relaxed exciton delocalization length of 4 ± 1 BChl molecules, independent of temperature, was obtained.320 Using multilevel Redfield theory including both static and dynamic disorder (exciton–vibrational coupling), exciton dynamics of B850 was investigated.258 The exciton size was found to be time dependent, delocalized over a large part of the B850 ring at very early times and have a relaxed size of ∼4 BChl molecules, again confirming this value as the relaxed exciton size of the B850 exciton. Super-radiance, or radiative rate, is a third method that has been used to access the exciton delocalization of B850 and LH1, and a very similar value, a few BChls, as in the analysis of transient absorption spectra was obtained.281 Together, these results show that the relaxed exciton size in B850 and LH1 is ∼4 BChl a molecules, largely independent of temperature. The exciton delocalization is time dependent and at very early times depending on excitation conditions it may span most of the B850 and LH1 rings.258 

The exciton within the B850 and LH1 rings is of course a dynamic feature, governed by BChl–BChl interactions and fluctuations of the site energies. Like for the B800 ring, energy transfer within B850 and LH1 has been studied by ultrafast transient absorption,320–323 fluorescence299,324 and 2DES325 measurements of isotropic and anisotropic signals, as well as photon echo methods.326 A large number of studies have been performed and although the precise numbers vary somewhat between the different measurements there is consensus regarding the main features of the energy transfer and the picture is very similar for both LH2 and LH1. Isolated complexes as well as membrane preparations of several different bacteria have been studied, without pronounced species or preparation type dependence. Following excitation, all studies agree that at room temperature there is a fast 50–100 fs decay of anisotropy and a very similar spectral relaxation (red-shift). In most studies a slower process, ranging from approximately one to a few ps is also reported in both anisotropy and isotropic decays. At low temperature (77 K and 4 K), the femtosecond process remains virtually unchanged, but the slower process becomes slower, particularly in the red part of the spectrum.320 The energy transfer dynamics of the B850 and LH1 BChl rings have been modeled by several authors, and found to be described by an ∼100 fs energy hopping time in a spectrally inhomogeneous pigment system. It is interesting to note that energy hopping between monomeric BChls was not found to give a proper description of the dynamics;320 energy hopping between dimeric sites was used in several studies,299,324 which may be seen as a result of the relaxed exciton size.

Interestingly, a recent anisotropy 2DES study of B850 at 77 K found a temporal mismatch of a few hundred femtoseconds between energy relaxation and depolarization, with the latter substantially slower.325 It was suggested that energy relaxation can happen locally without spatial motion, which appears to be hindered at cryogenic temperatures, especially at the low energy side. Whereas low mobility was explained by the presence of substantial energetic disorder in the B850 ring, the mechanism of the local relaxation remains to be identified.

Finally, coherent oscillations were also reported for B850 and LH1; at low temperature, a strong oscillation at 105–110 cm−1 and damping time of several hundred femtoseconds were observed, as well as weaker oscillations of higher and lower frequency. The oscillations persisted up to room temperature, albeit with reduced amplitude.82,323,324,327 In contrast to similar observations for the FMO protein,96 the oscillations were assigned to vibronic coherence without attributing a significant role in light-harvesting function.

d. Carotenoid to BChl energy transfer

In addition to BChl a, other key pigments in purple bacterial light-harvesting complexes are carotenoids. Their contribution to the absorption spectrum of LH1/LH2 fills the gap between the BChl a Soret and Qx/Qy bands, pointing to their importance for light harvesting. Indeed, fluorescence excitation spectra reported more than 40 years ago95,328 provided clear evidence of their role as light-harvesting pigments in LH2 and LH1 complexes. The first attempts to resolve the carotenoid-BChl energy transfer rates were reported by Gillbro et al.,329 but the data were limited by picosecond time resolution, which, as was shown later, was not sufficient to precisely determine the energy transfer rates. The first application of ultrafast spectroscopy with femtosecond time-resolution to study carotenoid-BChl energy transfer was carried out on the LH2 complex of R. acidophilus, revealing sub-picosecond rates for both Car-B800 and Car-B850 energy transfer.330 

These initial studies paved the way to a number of subsequent systematic studies, which, together with significantly improved instrumentation at the end of last century, revealed details about the energy transfer pathways involving carotenoids in purple bacterial antennae. It is well known that purple bacteria accommodate a large variety of carotenoids having conjugation lengths from 9 (e.g., neurosporene in the G1C strain of Rb. sphaeroides) to 13 (spirilloxanthin in Rhodospirillum rubrum). The conjugation length of a carotenoid is the key parameter determining the efficiency and pathways of carotenoid-BChl energy transfer. Reconstitution of LH2 from Rb. spheroides with spheroidene analogs having varying conjugation lengths revealed that prolongation of the conjugation length decreases the efficiency of carotenoid-BChl energy transfer.331 This observation was further corroborated by studies of various LH2's binding different carotenoids. Thus, shorter carotenoids, such as neurosporene, transfer energy with nearly 100% efficiency, while the long ones, such as spirilloxanthin, barely reach 30% efficiency.151,332

The crucial distinction is the efficiency of energy transfer via the carotenoid S1 state. While the S2 pathway has an efficiency in the 25%–40% range for essentially all LH2 and LH1 complexes, the S1 pathway is active only for carotenoids with the S1 state energy high enough to allow transfer to the Qy state of either B800 or B850. The breaking point is between N = 10 and N = 11, as spheroidene (N = 10), has an active S1 pathway with ∼80% efficiency, while lycopene or rhodopin (N = 11) exhibit essentially no transfer via the S1 pathway.332–335 These experimental results were also supported by rapidly developing computational techniques that were able to calculate carotenoid–BChl interaction and to reproduce reasonably the observed energy transfer rates.336,337

The picture that emerged from a number of studies at the turn of the century was, however, slightly complicated by experimental evidence of other spectral features that were unexplainable within the simple framework of two carotenoid excited states, S1 and S2, donating energy to two BChl states, Qx and Qy. First, transient absorption experiments extended to the near-IR region revealed a minor depopulation pathway from the excited S2 state, leading to formation of a carotenoid cation radical.335,338 The charge separation occurs between carotenoid and B800 BChl a and lasts for a few picoseconds. A possible functional role of this mechanism has never been elucidated. The efficiency of the radical formation is higher for shorter carotenoids, reaching about 10% for neurosporene and is nearly absent for carotenoids with N > 10.338 Furthermore, the enigmatic carotenoid S* state (see Sec. III A 2 b) was identified in LH2 and even proposed to serve as a minor energy donor in carotenoid-BChl energy transfer.213 The peculiar dependence of the S* signal amplitude on excitation intensity led to a proposal of complicated relaxation and energy transfer schemes,205 but it was shown later that the source of the S* signal in LH2 could be due to an electrochromic shift of the carotenoid absorption band.216 The electrochromic shift originates from the local electric field generated by the excited BChl nearby.339 Niedzwiedzki et al.216 showed that if this effect is taken into account, transient absorption spectra of LH2 complexes can be fully explained without invoking the S* state.

3. Energy transfer and trapping in the PSU

Having examined the intra-complex dynamics of LH2 and LH1, we are now prepared to explore the inter-complex energy transfer dynamics and the flow of energy through the antenna network toward the RC where it is trapped through charge separation, forming a transmembrane potential. Purple bacterial membranes have been imaged with atomic force microscopy at a resolution of ∼10 Å,340–342 showing LH1-RC complexes surrounded by a sea of LH2 complexes (Fig. 9). From this organization of the PSU, we expect energy transfer first between the LH2 complexes, then from LH2 to LH1, followed by a final transfer step from LH1 to the special pair in the RC. Studies of these processes of course require the presence of the complete PSU, or at least the relevant parts. The experiments have, therefore, been performed on membranes or whole cell preparations of several wild-type or mutant purple bacteria species.

Early pre-LH2-structure picosecond absorption studies reported a LH2-LH1 energy equilibration time of 37 ps (Ref. 293) and, with time-resolved fluorescence, two transfer times to LH1, 10 and 50 ps, were reported for LH2 complexes in close contact and more distant, respectively.343 Later, in transient absorption experiments with femtosecond resolution a fast ∼5 ps B850 to LH1 energy transfer time was resolved,297,344 as well as a slower 26 ps component.344 The fast ∼5 ps component was interpreted as energy hopping from a LH2 complex directly associated with a LH1 complex, and the longer 26 ps transfer time was interpreted as energy migration in the LH2 pool prior to transfer to LH1. In relation to the purple bacteria PSU, all these results taken together, suggest that direct LH2-LH1 ring-to-ring transfer occurs with an ∼5 ps transfer time and energy diffusion over the network of LH2 rings prior to hopping over to the LH1 ring takes 30–50 ps. The latter B850 diffusion time could be imagined to depend on the extent of the LH2 peripheral antenna (i.e., number of LH2s per LH1-RC), which probably varies between purple bacterial species and growth conditions.

With an overall energy transfer time of approximately 50 ps from the moment of light absorption in the peripheral antenna until energy is deposited in the LH1 ring of BChls in the vicinity of the RC, light harvesting in purple bacteria up to this point has nearly 100% quantum efficiency. It now remains to see how energy enters the reaction center and initiates charge transfer from the BChl special pair (P870) to the monomeric BChl primary electron acceptor. Several time-resolved absorption and fluorescence studies of a few different purple bacterial species, with excitation of the LH1 core antenna, have shown that energy transfer to the special pair occurs with a characteristic time of 40–50 ps.31,345–348 Knowing that the primary charge separation in the reaction center takes about 3 ps, it was concluded that the overall rate of energy trapping by the RC is limited by LH1 to P870 energy transfer, rather than the primary charge separation in the RC.31,345–348 This was confirmed by studies of purple bacterial membranes of RC mutants having a slowed down charge separation—a wide variation of CT times had only little effect on the overall trapping time.349,350 Traditionally, two types of trapping kinetics have been identified, “diffusion limited” and trap limited, respectively, where energy diffusion through the antenna network, or charge separation in the RC are rate limiting for the overall trapping rate. Distinct from either of these limiting cases, trapping in purple non-sulfur bacteria was termed “transfer-to-trap-limited.”347 The various energy transfer times characterizing the light-harvesting processes in purple bacteria are summarized in Fig. 10.281 

Based on AFM pictures of the purple bacterial membrane (Fig. 9) and the PSU model in Fig. 10, it can be deduced that the distance from BChls in the LH1 ring to P870 is the largest energy donor-acceptor distance in the whole PSU. It was realized that this distance, limited by the size of the RC protein and shape of the LH1 core antenna, is the reason for the relatively slow LH1-P870 energy transfer.281 It was further speculated that this large distance is necessary to avoid oxidation of LH1 BChls by the oxidized special pair; if this would happen, an oxidized BChl molecule in LH1 would be a very efficient quencher of excitation energy.281 The slight uphill energy transfer condition (going from B875 in LH1 to P870 in RC)351 most likely serves as a protection mechanism, where energy can be easily detrapped from the closed RC under high light conditions.

What ultrafast spectroscopy taught us about photosynthetic purple bacteria

Ultrafast spectroscopy has given us a detailed picture of the energy flow through the photosynthetic unit of purple bacteria, from the ultrafast, local, inter-chromophore energy transfer within the LH1 and LH2 antenna proteins, to more long-range transfer between the antenna proteins and antenna to RC.

  • The peripheral LH2 antenna exhibits energy transfer on two time scales—ultrafast sub-ps migration of excitons delocalized over a few (∼4) BChl molecules within the ring of strongly coupled B850 molecules, and slower, ∼1 ps, transfer from localized B800 excitons to B850, as well as energy migration among the B800 molecules.

  • Energy dynamics within the BChls of LH1 is similar to that of B850.

  • Ring to ring LH2-LH1 energy transfer occurs with a characteristic time of ∼5 ps, and transfer over several rings takes more time, a few tens of ps.

  • The final energy transfer step from the LH1 ring to the RC is the slowest step in the overall light-harvesting process, ∼35 ps.

  • Carotenoids collect blue and green light and transfer it with high efficiency on the sub-ps to ps timescale to the BChls of LH2 and LH1.

  • All these individual energy transfer steps add up to an overall antenna-to-RC energy transfer time of ∼60 ps for photochemically active RCs, resulting in very efficient conversion of absorbed photons to separated charges in the RC.

1. Light harvesting in photosystem II

In plants, photosynthesis takes place in chloroplasts with the help of chlorophyll and carotenoid molecules organized in two photosystems, photosystem I (PSI) and photosystem II (PSII). The light-harvesting machinery of PSII, situated in the grana membranes of the chloroplasts, consists of several different LHCs feeding excited state energy into the RCs they surround. A PSII supercomplex, called C2S2M2, has been isolated and structurally characterized from several species at various levels of resolution.352–354 It is a dimer complex, the one from higher plants contains 326 pigment molecules bound to a protein assembly consisting of LHCs surrounding two RCs.352 Each monomer consists of two light-harvesting complexes II (LHCIIs), three minor LHCs (CP24, CP26, and CP29), two core antenna complexes (CP43, CP47), and one RC. Energy transfer has been studied in all the individual, separately isolated, complexes, as well as various membrane preparations, which may be expected to hold multiple copies of interconnected C2S2M2 supercomplexes.355 We will discuss the results for the various levels of organization with the aim to obtain a picture of the energy flow through the PSII light-harvesting network of pigment–protein complexes.

a. Structural aspects of PSII LHCs

Before we discuss energy transfer in PSII LHCs, we give a short account of their structures. LHCII is the major peripheral light-harvesting complex (Fig. 11), present as a trimer in chloroplasts, binding ∼50% of all chlorophylls in green plants. Its three-dimensional structure was first determined by electron diffraction on two-dimensional crystals, initially to a resolution of 6 Å (Ref. 356) and three years later to 3.4 Å.357 The LHCII structures based on two-dimensional crystals did not have sufficient resolution to distinguish between Chl a and Chl b molecules, or to establish the direction of their x and y axes. However, all this was achieved with the structure obtained later to 2.72 Å resolution with x-ray crystallography,358 providing unambiguous determination of the identities and positions of 8 Chl a and 6 Chl b molecules. In addition, four carotenoid molecules were identified, two luteins and one neoxanthin and violaxanthin each. CP24, CP26, and CP29 are smaller peripheral LHCs present in monomeric form in the photosynthetic membrane (Fig. 11). Early structural models of CP24 indicated binding of five Chl a and five Chl b molecules, as well as two carotenoids, lutein and violaxanthin.359 A more recent high-resolution structure,360 however, shows that the complex binds six Chls a, five Chls b, and one each of lutein, violaxanthin and β-carotene. Based on the sequence homology with LHCII, both CP26 and CP29 were believed to bind 8–9 Chl molecules with a Chl a/Chl b ratio of ∼2, and two carotenoid molecules.361 However, more recently the high resolution crystal structures of both CP26362,363 and CP29363,364 have been determined. These studies show that both complexes bind 13 Chls; CP 26—nine Chls a and four Chls b, and CP29—ten Chls a and three Chls b. Both complexes hold three carotenoid molecules (CP26: two luteins and one neoxanthin; CP29: one lutein, neoxanthin, and violaxanthin). The protein frameworks in the earlier structural models of all three complexes are similar to the actual structures and the estimated Chl a/Chl b ratio is approximately correct, but the number of bound Chls is underestimated. CP43, with 13 Chls, and CP47, with 16 Chls, and both with 2–3 β-carotenes, constitute the core antenna of the PSII photosynthetic unit,354,363,365,366 relaying the excitation energy from the peripheral antenna to the RC (Fig. 11). Spectroscopic studies prior to availability of high-resolution structures were analyzed in terms of the earlier structural models and, as we will see below from our discussion on energy transfer, they often aimed at providing complementary information to the structural models (like Chl identity, a or b, and direction of transition dipole moments).

b. Energy transfer in the peripheral light-harvesting complexes of PSII

Energy transfer and trapping in PSII preparations have been studied with time-resolved methods since the beginning of the 1980s. Much of the early work was reviewed by van Grondelle and colleagues in 1994.94 As a starting point of our discussion of more recent work on each light-harvesting complex, we will briefly summarize the level of understanding of light harvesting and energy transfer around 1994 (for details we refer to Ref. 94).

c. LHCII energy transfer

The early, pre-1994, work on LHCII includes many picosecond fluorescence and transient absorption studies, and a few investigations with sub-ps resolution. The observed energy transfer was characterized by multi-exponential kinetics with time constants ranging from hundreds of femtoseconds to several tens, or even hundreds of picoseconds, while recorded nanosecond lifetimes reflected the intrinsic excited state lifetimes of Chls. All these measurements were performed before the first crystal structure of LHCII was published,357 which means that detailed structure-dynamics-function correlations were not possible. Nevertheless, Chl b to Chl a energy transfer was generally reported to be very fast, few-hundred of fs, and slower times from a few ps to several hundred ps were often assigned to energy equilibration between various (non-specified) Chl a pools, or between LHCII-monomers if the investigated samples were trimeric LHCII. As time-resolved spectroscopy techniques developed, time resolution improved and crystal structure became available, in particular, the high resolution 2.72 Å structure,358 an increasingly more detailed picture of the energy flow through LHCII and its connectivity to other PSII components has been obtained. We will illustrate how the present picture has developed with the help of several selected works on LHCII.

An important step was taken by several authors when measured rates were compared with calculated energy transfer rates based on the Kuhlbrandt LHCII structure.357 Visser et al. performed sub-ps transient absorption measurements on the LHCII trimer at 77 K.367 Excitation was performed at several wavelengths in the range 649–682 nm, providing selective excitation of both Chls b and Chls a, and transient spectra were measured in the 630–700 nm range. Chl b → Chl a energy transfer was concluded to occur with three time constants, <0.3, 0.6, and 4–9 ps, and energy transfer among the Chl a molecules was reported to proceed with similar transfer times, 0.4, 2.4, and 10–20 ps. The comparison to calculated rates based on the LHCII structure357 led to some correlation between measured rates and Chls in the structure. It was also concluded that the wide range of observed energy transfer times, from <0.3 to ∼20 ps, most likely reflects the irregular arrangement of the pigments in the complex, with much less symmetry than LH2 of purple bacteria. A similar approach was taken in Ref. 368, and very similar Chl b → Chl a transfer times (175, 625, and 5 ps) were obtained and analyzed with a kinetic model based on the structure.357 Chl b → Chl a energy transfer in monomeric LHCII369 was found to be considerably slower than in trimeric LHCII, and it was argued that the reason is loss of accepting Chl a molecules in the monomer. Three-pulse photon echo peak shift (3PEPS) and transient grating (TG) measurements were used to provide additional details to the scheme of energy transfer pathways in LHCII.370 The TG measurements confirmed the Chl b → Chl a transfer timescale reported in earlier transient absorption measurements,367–369 i.e., 300 fs and 2.8 ps. Similarly, transfer from blue- to red-absorbing Chls a was found to be characterized by the time constants 230 fs and 6 ps. The 3PEPS measurements were concluded to reflect energy equilibration among Chls of similar excited state energy and found to occur with time constants of ∼150 fs and ∼1 ps for both Chl a and Chl b. From detailed analysis of the measured kinetics it was considered likely that mixed Chl sites exist in LHCII, but no support for this appears to be found in the structural and assembly analysis of the C2S2M2-type PSII-LHC supercomplex.360 

As a next step in the analysis of LHCII energy transfer, an exciton model was proposed to explain the results of several different steady state (absorption, linear-dichroism, and super-radiance) and ultrafast (transient absorption, 3PEPS, and TG) measurements.371 The nonlinear response was calculated using the Liouville equation for the density matrix with the Redfield relaxation superoperator in the exciton eigenstate basis. A few configurations of Chls b and Chls a, with the Kuhlbrandt structure357 as a starting point, assigning Chl b (a) identities, site energies, and dipole moment orientations, were found, which allowed simultaneous fits of all data and returned timescales for the energy transfer dynamics. Energy relaxation within the Chl b and Chl a pools of pigments was concluded to include 250–600 fs exciton relaxation processes, 600–800 fs hopping between spatially separated pigment clusters and picosecond timescale migration between localized states. Chl b → Chl a transfer was found to proceed along a very fast, 120 fs, channel. Within Chls a, a delocalization over 1.4–1.8 monomers was found at room temperature.

With the high-resolution structural model of Liu et al.358 available, the energy transfer dynamics and steady state spectra of LHCII were modeled again.372 An exciton model of the LHCII trimer was used to provide a simultaneous fit to the steady state spectra (absorption, linear dichroism, fluorescence and transient absorption kinetics. Following excitation of Chl b at 650 nm, a very rapid, few-100 fs energy transfer to two specific Chls were observed, b605 and a604 (Fig. 12), followed by a few-ps focusing of the energy to a monomeric Chl a, a604, and finally even slower 10–20 ps concentration of the energy to a cluster of Chls a—a610, a611, a612, with emphasis on a610. The energy equilibration within and between Chls a clusters was seen to proceed on the few to several-100 fs timescale. A similar situation holds for Chl b. It was speculated that final acceptor Chls a, located at the outer side of the LHCII monomer, provides good connection to other PSII subunits.

The 2DES results on LHCII by Schlau-Cohen et al.,373 summarized in Fig. 13, basically confirmed earlier results372 and showed sub-100 fs relaxation through spatially overlapping states, several hundred femtosecond transfer between nearby chlorophylls, and picosecond energy transfer across the membrane, from the luminal to stromal side. At long times, all energy was observed to concentrate into the a610–a611–a612 Chls a cluster. In previous modeling of steady state and time-resolved data (see, e.g., Ref. 371), electronic transition energies of the Chls were part of the modeling.

More recent 2DES work on LHCII trimers at room temperature found direct and multistep cascading pathways from the high-energy chlorophyll b states to the lowest-energy chlorophyll a on the time scale from hundreds of fs to tens of ps.374 Insights into the energy transfer dynamics and mechanisms obtained with the help of 2DES have been recently reviewed in Ref. 375.

d. Energy transfer in the minor, monomeric, LHCs CP24, 26, 29

As illustrated in Fig. 11, CP24, CP26, and CP29 are monomeric light-harvesting complexes in PSII located between the main peripheral LHCII antenna and the RC core complex. The three complexes together bind ∼15% of the total Chl content of PSII and are therefore believed to play a minor role in light harvesting, but they may have important function in regulating the energy flow from LHCII to the PSII core.376,377 The energy transfer dynamics in these minor light-harvesting complexes is much less studied than in LHCII. Nevertheless, several time-resolved studies have been performed, and we will briefly summarize the development leading to the present picture of light harvesting by the minor LHCs.

Energy transfer among the Chl molecules in CP29 was studied using transient absorption spectroscopy at 77 K.378 Since a high-resolution structure of the complex was not available at the time of the study, spectral and transition dipole moment direction assignment of the pigment molecules was performed using the protein sequence homology with LHCII, as well as time-resolved spectroscopy and linear-dichroism data. Based on this assignment, the measured kinetics were modeled with Förster energy transfer theory. Ultrafast, ∼0.3 ps, as well as slower, ∼2 ps, Chl b → Chl a transfer was concluded, in addition to Chl a equilibration similar to that previously observed for LHCII, ∼0.2 and 10–20 ps. These results were further substantiated in Ref. 379. Here, building on the LHCII structure357 and a previous determination of site energies and transition moment directions,380 a more detailed structural model of CP29 was constructed. Very good agreement between the two models was obtained. The energy transfer rates between chlorophylls at room temperature were calculated using Förster theory and compared to ultrafast measurements. Figure 14 summarizes the results of the kinetic model indicating bidirectional excitation transfer over all CP29 Chls a species, with maximum rates >10 ps−1; Chl b → Chl a transfer in the sub-ps range was also estimated, in good agreement with Ref. 378.

3PEPS and TG measurements were used to further elaborate the energy transfer dynamics in CP29.370 The complex was excited in the Chl b band at 650 nm and in the blue shoulder of the Chl a band at 670 nm. The TG measurements with 650 nm excitation probed Chl b → Chl a energy transfer and was found to occur with the time constants 130 fs and 2.2 ps. Excitation at 670 nm probed transfer from blue- to red-absorbing Chls a and was found to be characterized by the time constants 300 fs and 5 ps. The time constants from the 3PEPS measurements were quite different from those of the TG measurements and concluded to reflect energy equilibration among Chls of similar excited state energy. For Chl b, this was found to occur with time constants of 360 fs and 3 ps. Equilibration among relatively blue Chls a was concluded to proceed with similar times as for Chl b, 3 ps. From detailed analysis of the measured kinetics, it was deduced that mixed Chl sites exist in the CP29 complex. The same conclusion was drawn in a detailed transient absorption study of Chl b → Chl a transfer,381 based on the observation of four different lifetimes, 150 fs, 600–800 fs, 1.2, 5–6 ps, for the Chl b → Chl a process, considering that CP29 at this time was believed to have only two sites with exclusive Chl b binding. With the presently accepted number of three Chls b in CP29, the conclusion, nevertheless, appears correct on the basis of the time-resolved spectroscopy results. However, little support for this claim appears from the structure and assembly analysis of the C2S2M2-type PSII-LHC supercomplex.360 The TG and 3PEPS measurements confirmed earlier transient absorption measurements of Chl b → Chl a and downhill Chl a energy transfer,378,379 and added information about the timescale of energy equilibration among Chls b and Chls a of similar energy.

The 2D electronic spectroscopy study of Fleming and co-workers,382 estimating the relative angle between electronic transition dipole moments of energy transfer chlorophyll pairs in CP29, is another example of how spectroscopy was used to add missing structural information to structural models with low resolution. It was also discussed how selected pairs could play the role as entry or exit points from LHCII or the core antenna, respectively, thus providing information about possible connectivity between the pigment–protein complexes of the PSII supercomplex.

Energy transfer dynamics in CP24 and CP26 are even less studied than in CP29. A first fs transient absorption study was performed by Marin and co-workers383 on both complexes. The two complexes exhibit somewhat different kinetics. CP26 was found to be quite similar to LHCII with two lifetimes, ∼0.2 and 1.1 ps, assigned to Chl b → Chl a transfer, and multiexponential Chl a ↔ Chl a energy transfer and equilibration (1.5–3.5 and 10–15 ps). In CP24 most of the Chl b → Chl a transfer was characterized by a 0.6 ps time constant and Chl a ↔ Chl a transfer appeared to be somewhat slower than in CP26. The differences were discussed in terms of different spectral properties of CP24 as compared to CP26 and LHCII, i.e., CP24 being enriched in Chl b and a 670 nm spectral form of Chl a.

Energy relaxation processes have been studied also in the asymmetric trimer complex LHCII(M)−CP29−CP24 with 2DES, where M indicates that LHCII in moderately bound to the core complex.384 The study observed faster energy equilibration in the intermediate levels in this complex as compared to the LHCII trimer. The observations were supported by structure-based calculations, and the accelerated dynamics were attributed to the structural change of LHCII(M).384 

From this account of the energy transfer dynamics in the various LHCs of the peripheral antenna of PSII, it appears that they all share the broad dynamical features—femtosecond to few picoseconds energy transfer from Chls b to Chls a, followed by picosecond timescale energy transfer among Chl a molecules, such that energy is concentrated to the lowest-energy Chls a of each complex within ∼10–20 ps. With the organization of the PSII supercomplex in mind (Fig. 11), a superficial picture of the energy flow from the outer antenna toward the core suggests that it takes a few tens of ps until the energy reaches the PSII core antenna with its RC. We will next discuss the energy dynamics within the core complex, CP43 + CP47+ RC, and end with some results on the PSII supercomplex and intact membranes, to arrive at a picture of the energy flow through the entire PSII photosynthetic unit.

e. PSII core complex (CP43 + CP47 + RC) energy transfer

Early (<1994) picosecond transient absorption and fluorescence studies of the PSII core complex often reported biphasic decays of antenna excitations, with lifetimes 80–100 and ∼500 ps (see, e.g., Ref. 94 for a review). The results were generally interpreted in terms of the “exciton/radical pair equilibrium” model.94,385 In this model, it was assumed that the energy difference between the equilibrated excited state of the antenna and the primary radical pair state of the RC was sufficiently small to cause the observed biphasic decay of antenna excited states. The initial excitation equilibration between the antenna and the special pair (P680) was assumed to take less than 10 ps. The 80–100 ps decay would then correspond to the initial charge separation (i.e., formation of P+IQ (I = pheophytin; Q = quinone)), and the ∼500 ps component to charge stabilization (i.e., formation of P+IQ). In terms of the limiting models for antenna exciton decay used at this time, the energy dynamics of PSII would be characterized as “trap limited.”94 In the following text, we will discuss the results from time-resolved spectroscopy and theoretical simulations, supported by new insights into the organization of the PSII pigment system that change this early picture.

The first PSII structures386–388 opened up the possibility to more closely examine the interaction between the core antenna and RC pigments. The distance between the chlorophyll molecule in CP47 closest to the RC pigments, ∼21 Å, is much longer than the average intra-CP47 Chl–Chl distance (∼9 Å). Calculations389 based on the PSII structure suggested slow, ∼100-ps timescale, energy transfer from CP47 to the RC, in sharp contrast to the rapid, sub-ps, equilibration of excitation energy between antenna and RC pigments suggested from early picosecond measurements and the exciton/radical pair equilibrium model. More recent modeling of steady state and time-resolved optical spectra390 gave a similar result, and showed that decay of excited states in the PS-II core antenna with active RCs is limited by the energy transfer from the CP43 and CP47 complexes to the RC occurring with a time constant of 40 − 50 ps at room temperature. Another interesting result of these calculations is that with QA of the RC in its reduced state, PSII is predicted to switch into a photoprotective state by reverse energy transfer from the RC into the antenna.

The CP47 → RC energy transfer was examined experimentally in transient absorption and time-resolved fluorescence experiments391 and it was concluded that there is no fast equilibration of excitation energy between antenna and RC pigments, in agreement with the calculations mentioned above.389,390 Thus, both experiment and calculations389–391 appeared to contradict the previously accepted exciton/radical pair equilibrium picture of excitation trapping in PSII and point to slow, much slower than primary charge separation, energy transfer from antenna to RC. Several more works provided additional insights into this matter, and support to the first indications of slow CP43/CP47 → RC energy transfer. This was performed for PSII core complexes from green plants392 and the cyanobacterium Synechocystis sp. PCC 6803,393 using femtosecond visible/mid-IR pump-probe spectroscopy. Very similar results were obtained for the complexes of both plants and cyanobacteria, and a kinetic model was constructed (Fig. 15), which, in addition to providing time constants for charge separation and stabilization within the RC, showed that energy transfer from the CP43 and CP47 antenna complexes to the RC is slow and characterized by 30–40 ps time constants.

In a more recent study on the PSII core complex, time-resolved fluorescence measurements in the temperature range of 5–180 K were compared with energy transfer simulations using Redfield theory for intra-domain exciton transfer and modified Förster theory for inter-domain transfer.366 Two low energy emitters were identified C685 at ∼685 nm and C695 at ∼695 nm (Fig. 16) and rate constants were obtained for the energy flow within CP43 and CP47, as well as from the antenna complexes to the RC (Fig. 16). C685 was identified with Chl45 or Chl43 in CP43 (red pigments in Fig. 16; Chl numbering according to Ref. 394) and C695 with Chl29 in CP47. C685 was suggested to have the role of a conduit of energy to the primary donor in the RC, and Chl29 as an energy acceptor from the peripheral antenna CP29. Also here, energy transfer from the core antennae to the RC was concluded to proceed with tens of ps time constants. The PSII core complex was also studied with 2D electronic spectroscopy at 77 K.395 A global analysis of the high time-resolution 2D data shows rapid, sub-100 fs energy transfer, as well as 2D spectral signatures of slower energy equilibration processes occurring on several to hundreds of picosecond timescales. These slow rates (at 77 K) are similar to those observed in Ref. 366 and illustrated in Fig. 16. At room temperature these slow energy transfer steps will be much faster, probably on the sup-ps to few-ps timescale, as indicated in other studies on isolated CP43 and CP47 (see below and Refs. 396 and 397).

Intra-CP43/CP47 energy transfer has also been studied in isolated CP43 and CP47 complexes. Transient absorption and time-resolved fluorescence spectroscopy experiments at 77 K (Ref. 396) showed that spectral relaxation occurred on two timescales, 0.2–0.4 and 2–3 ps in both complexes. Later, a combination of TCSPC and streak camera fluorescence measurements397 at room temperature showed that excitation energy transfer between blue and red states in both antenna complexes is dominated by sub-picosecond processes and is completed in less than 2 ps. From both works it was concluded that the ultrafast intra-antenna energy transfer steps do not represent a bottleneck in the rate of the primary processes in intact PSII.

f. Carotenoid-to-chlorophyll energy transfer in the plant PSII antenna

All antenna complexes associated with PSII contain carotenoids, which have a twofold role in these complexes. They act as light-harvesting pigments, but they also serve as important photoprotective agents (see Sec. III A 2). The light-harvesting function of carotenoids relies on their ability to transfer energy to chlorophylls. The first ultrafast spectroscopy studies addressing this question appeared in 1997 and provided controversial results on carotenoid-Chl energy transfer in LHCII. Peterman et al.398 suggested fast, ∼220 fs, energy transfer from carotenoid to Chl a, while an even faster (sub-200 fs) transfer channel was reported by Connelly et al.,399 but with Chl b as the main energy acceptor. Later studies confirmed the ultrafast energy transfer channel via the carotenoid S2 state, occurring with an ∼100 fs time constant to compete with the fast S2–S1 relaxation of carotenoids.400–403 LHCII contains two luteins, neoxanthin and violaxanthin.358 Transient absorption spectroscopy identified Chl b as the energy acceptor for energy transfer from neoxanthin, while Chl a accepts energy from violaxanthin and lutein.400,401 Fluorescence upconversion data corroborated these findings, demonstrating an efficient (50%–60%) S2-mediated energy transfer pathway in LHCII, yet the neoxanthin S2 channel was considered to be only minor.402,403 An efficient S2 channel associated with lutein has been identified from 2DES measurements, however transferring energy to Chl a via an intermediate carotenoid Sx state.404 

The first studies did not detect any slower channel attributable to energy transfer via the carotenoid S1 state. Such a pathway was, however, identified later and assigned to either lutein-Chl,400 or neoxanthin-Chl401 energy transfer. The time constant associated with the S1 route was around 1 ps, though identification of individual carotenoids to this pathway was problematic. As mentioned, either lutein or neoxanthin were suggested to be the donors in the S1 route, with efficiency not exceeding 20%.400,401 A different picture of energy transfer pathways was obtained from two-photon excitation experiments that could presumably excite the carotenoid S1 state directly.405 However, two recent studies demonstrated that two-photon excitation experiments suffer from a significant contribution of Chl a/Chl b,406,407 challenging the original data interpretation. Thus, the 250 fs rise of the Chl a signal reported in Walla et al.405 could be at least partly associated with Chl b to Chl a transfer, or to relaxation within the Chl a pool. On the other hand, broadband 2DES data reported recently also detected an ∼300 fs energy transfer channel, but via the elusive Sx state whose origin is still unclear.404 This carotenoid excited state lies below the S2 state, but higher than the S1 state, implying that the two-photon excitation experiments might possibly see this channel by exciting the Sx state by two-photon excitation.408 However, recent experiments employing femtosecond Raman spectroscopy did not reveal any sub-ps energy transfer channel via the S1 route, nor did they identify any Sx state. Instead, an ∼7 ps energy transfer channel from the lutein S1 state was reported.409 It shortens the S1 lifetime of lutein to ∼4.4 ps, which is very close to the lutein S1 lifetime of 3.4 ps reported in one of the first ultrafast spectroscopy studies targeting the carotenoid-Chl energy transfer in LHCII.401 

Energy transfer between carotenoids and chlorophylls has been studied also for the minor antenna complexes. CP29 binds three carotenoids, lutein, violaxanthin and neoxanthin and the overall picture of carotenoid-Chl energy transfer is similar to that in LHCII. A ∼100 fs S2 channel with >60% efficiency was reported in the first ultrafast spectroscopy study of CP29.400 Later, in addition to the S2 channel, two other energy transfer channels associated with hot S1 (∼700 fs) and relaxed S1 (∼10 ps) state were suggested.381 However, as shown recently, the sub-ps dynamics is at least partly due to Chl b to Chl a energy transfer that is not easily separable from the carotenoid-Chl energy transfer.214 This study has also shown that the carotenoid S1 lifetime in CP29 (13 ps) is not associated with changes in the Chl a bleaching signal, implying that essentially no energy is transferred via the S1 route. Thus, only the S2-mediated transfer plays a substantial role in CP29. A very similar pattern of energy transfer pathways was reported for CP26 and CP24. The dominant carotenoid-Chl energy transfer proceeds via the S2 pathway, possibly with a minor contribution from a route involving a hot S1 state.383 However, a possible mixing of this hypothetical route with Chl b to Chl a energy transfer may occur. Transfer from a relaxed S1 state has not been reported for any of the minor complexes. Carotenoid-Chl energy transfer in the antenna complexes associated with the PSII core, CP43 and CP47, was studied by fluorescence upconversion. In contrast to other PSII antennas CP43 and CP47 bind exclusively β-carotene, which transfers energy to Chl a via the S2 state with ∼30% efficiency. No S1 channel was detected in these complexes.410 

g. Energy transfer in intact PSII

Energy transfer and excitation trapping in intact PSII has been studied since the mid-1980s by many different authors. Much of this work, including PSII in a variety of conditions, e.g., open/closed reaction centers, absence/presence of multivalent cations, the phosphorylation state of LHCII, etc., was performed during the first ten years of this research and reviewed in 1994.94 In one way, the results could be said to be remarkably consistent exhibiting three groups of decay components, 50–100 ps, 0.4–1.0 ns and 1.2–2.2 ns.94 On the other hand, considering the spread of lifetimes within each group and the complexity of the PSII pigment system as we know it now, it is questionable, as we will see below, that mechanistic detail can be concluded from this type of data. Nevertheless, the early multi-exponential kinetics of PSII excitations were lively discussed in terms of the two limiting models for excitation decay—diffusion limited and trap limited, and various modifications of these models (this is reviewed in detail in Ref. 94). The exciton/radical pair equilibrium model385—a variant of trap limited kinetics—gained some acceptance not only for the smaller PSII core complex (see above), but also for intact PSII. Within this model bi-exponential excitation decays of PSII preparations with open (photochemically active) RCs were interpreted analogously to the kinetics of the PSII core complex411—∼300 ps was assigned to initial charge separation (i.e., formation of P+IQ), and 550–650 ps to charge stabilization (i.e., formation of P+IQ); the longer time for charge separation in intact PSII, as compared to the PSII core complex, is a result of exciton diffusion over the larger antenna of the complete PSII. To explain the experimental data, heterogeneity of PSII (i.e., two different RCs, α and β, with different charge separation rate) had to be assumed.411 

Time-resolved fluorescence measurements on PSII-containing membranes (BBY particles) from spinach with open reaction centers412 constitute an early step away from the exciton/radical pair equilibrium model for intact PSII. The measured decay kinetics exhibited an average decay time of 150 ps, and a coarse-grained analysis method to model the energy migration and charge separation processes, based on an early version of the PSII supercomplex structure,352,355 suggested that excitation diffusion within the antenna contributes significantly (with a migration time of ∼100 ps) to the overall charge separation time in PSII.

A detailed theoretical analysis of energy transfer and trapping in PSII was performed on the PSII supercomplex,352,353 using a generalized Förster/modified Redfield rate equation approach.413 This PSII supercomplex, called C2S2M2, is a dimer complex containing 326 pigment molecules bound to a protein assembly consisting of light-harvesting antenna complexes surrounding two RCs. Each monomer consists of two LHCIIs, three minor LHCs (CP24, CP26, and CP29), two core antenna complexes (CP43 and CP47) and one RC. The calculations showed that the kinetics of light harvesting cannot be simplified to a single rate limiting step. Instead, substantial contributions arise from both excitation diffusion through the antenna pigments and transfer from the antenna to the reaction center, where charge separation occurs. The characteristic times for these processes were found to be approximately equal, 110 and 100 ps, respectively. It was concluded that because of the lack of a rate-limiting step, fitting kinetic models to fluorescence lifetime data cannot be used to derive mechanistic insight on light harvesting in PSII, in contrast to the multiple early studies summarized in Ref. 94.

The energy transfer and trapping were also studied theoretically in a sub-system of the PSII supercomplex, containing one LHCII, one CP43, and a RC, holding 33 pigment molecules, using a non-Markovian quantum master equation approach.414 The results were quite similar to those in Ref. 413, showing that energy diffusion through the antenna takes several tens of ps, and transfer from the core antenna to the RC proceeds on a similar timescale. In terms of the limiting cases often used to describe excitation dynamics in photosynthetic systems, the energy transfer and trapping in the PSII supercomplex413,414 can be described as a hybrid between diffusion limited and transfer-to-trap limited dynamics.281,347

What ultrafast spectroscopy taught us about energy transfer and trapping in PSII

  • Energy transfer among Chls within individual light-harvesting complexes, LHCII, CP24, 26, 29, 43, and 47 occurs on the ∼100 fs to ∼tens of ps timescale, and within ∼10 ps the energy is concentrated to the lowest energy pigments/states of each complex.

  • Energy flows from the peripheral antenna, LHCII, CP24, and CP26 toward the core antenna CP43 and CP47. The pathways of energy flow are defined by bottleneck pigments/states in the energy donating and receiving complexes.

  • The total time for energy to flow from the peripheral antenna to the vicinity of the RC is at least several tens of ps and up to ∼100 ps. Since the time of each Chl–Chl energy transfer step is several orders of magnitude shorter than the Chl intrinsic excited state lifetime (2–3 ns), the overall energy transfer quantum efficiency is >90%.

  • Energy transfer from the CP43/47 core antenna to the RC is much slower, 30–40 ps, than primary charge separation in the RC (see Sec. III I 2 on ET in PSII RC), and there is no equilibrium established between excitations in the antenna and in the RC. This mode of energy trapping by the RC has been termed “transfer to trap limited” and is similar to that in non-sulfur purple bacteria.

  • In all plant PSII antenna complexes, carotenoids transfer energy to chlorophylls predominantly via the S2 state. To compete with the fast S2–S1 relaxation, the energy transfer occurs on an ∼100 fs timescales.

2. Light harvesting in photosystem I

a. Structural aspects of PSI

PSI is a large complex whose main role in photosynthesis is to transfer electrons from plastocyanin to ferredoxin. The electrons are then used to produce the hydrogen carrier NADPH, and ATP. The light-harvesting apparatus of PSI is organized differently from PSII. While PSII contains individual light-harvesting complexes assembled together with the RC in the C2S2M2 supercomplex (Sec. III E 1 a), in PSI both RC and light-harvesting pigments are bound to a single large complex denoted as PSI core. The first structure of the cyanobacterial (Thermosynechococcus elongatus) PSI core reported in 2001415 showed that it consists of a few subunits, but the pigments, 96 Chl a and 22 β-carotene molecules (five of which are cis-isomers), are predominantly bound to the PsaA and PsaB subunits. A similar structure of the PSI core has been reported for higher plants, though some minor differences in pigment binding have been identified.416 The main difference is that the PSI core from cyanobacteria forms trimers (some tetrameric cyanobacterial PSI complexes have also been reported),417,418 while higher plants feature a monomeric PSI core with accessory LHCI antenna complexes denoted as Lhca1–4.416,419,420 Monomeric PSI cores of different organisms exhibits some variations in the number of pigments, thus 98 Chl a molecules were identified in the PSI core from Pisum sativum,416 and 26 carotenoids were found in the monomeric PSI core from T. elongatus.421 The reader is referred to the above-mentioned papers for structural details.

PSI is probably the most efficient light-harvesting complex in nature, as it converts the absorbed photons to electrons with quantum efficiency close to one.422,423 This remarkable efficiency is achieved by the arrangement of pigments in PSI that are tightly packed to allow for efficient energy transfer, yet separated enough to prevent pigment concentration quenching.424 Thus, in PSI the distances, orientations and inter-pigment couplings are set to allow for nearly lossless energy transfer through the PSI energy landscape.423,425 The high light-harvesting capacity of PSI even initiated studies targeting the possibility of incorporation of PSI into photovoltaic devices.426,427 The high efficiency of PSI is even more remarkable considering that PSI of almost all organisms contains Chls absorbing at energies below the energy of the P700 primary donor.428 They are denoted as red Chls in PSI, are found in both cyanobacterial and plant PSI, and their emission bands may extend beyond 750 nm in some organisms.425,428 In the PSI-LHCI complex of plants, the red Chls are predominantly located in the accessory LHCI antenna, though some are likely located even in the PSI core.429 The presence of red Chls in the core is also evidenced by their occurrence in cyanobacterial PSI consisting only of the trimeric PSI-core. However, the loss of red chlorophylls upon monomerization of the trimeric cyanobacterial PSI core suggests their location at the interface between monomers,421,430 which cannot be the case in the monomeric PSI core of higher plants.

b. Energy transfer within the Chl pool in PSI

In contrast to LHCII (or other antenna complexes associated with PSII), the individual energy transfer pathways in the PSI core are extremely difficult to track due to the nearly 100 Chls associated with each PSI core monomer. The first ultrafast spectroscopy study addressing energy transfer in the PSI core used fluorescence upconversion depolarization measurement.431 This method revealed ∼200 fs anisotropy decay assigned to individual energy transfer steps between Chls within the PSI core. A slower, 5 ps isotropic component was assigned to equilibration within the PSI core. Comparable results were reported in subsequent studies, though the actual values varied slightly. The Chl–Chl energy transfer steps occurred on the 100–400 fs timescale,432 while the slower component was assigned to energy transfer/equilibration with the red Chls. This component varies with species, and values ranging from 2 to 10 ps were reported.432–436 

This picture of the energy flow through the PSI core has been also confirmed by 2DES used to follow the energy transfer in the cyanobacterial PSI core. Yet, this method suggested an additional fast (∼200 fs) energy equilibration process involving red Chls that is present in addition to the 2–3 ps equilibration revealed in earlier studies.437 Moreover, 2DES detected an ultrafast, ∼50 fs, equilibration time in the PSI core, which remained unidentified in earlier transient absorption studies.437,438 The overall trapping time in the PSI core, defined as delay between the absorption of a photon and the moment of charge separation, is 15–40 ps depending on species.423,425,437,439 These studies assume that the trapping time is migration-limited with the slowest step being the energy transfer from antenna to RC, while the charge separation process itself occurs with a time constant not exceeding 2 ps. It should be mentioned that modeling transient absorption data led to an alternative scenario assuming that charge-separation in the PSI core is trap-limited with much faster (∼8 ps) energy transfer from antenna Chls to RC.436,440

The outer LHCI antenna of higher plants consists of four proteins Lhca1–4, which resemble the LHCII or CP29 proteins from PSII.416 Each Lhca protein binds 13–14 Chls with a Chl a/Chl b ratio of ∼3.7 and three carotenoids. In contrast to LHCII/CP29, Lhca from PSI binds β-carotene in addition to lutein and violaxanthin. One extra lutein is located between Lhca1 and Lhca4, resulting in a total of 13 carotenoids bound to the Lhca antenna system of PSI.416 The Lhca antenna system accommodates most of the red chlorophylls in the PSI-LHCI complex, and they are predominantly found in Lhca3 and Lhca4.441 In Lhca4, the red chlorophyll states are proposed to result from mixing of CT states with excitonic states.442 

The first studies employing ultrafast spectroscopy to resolve the energy transfer network in Lhca complexes revealed sub-picosecond Chl b to Chl a energy transfer in Lhca1 and Lhca4, followed by 3–5 ps equilibration within the Chl a pool.433,443,444 Additional dynamics was recently identified via application of 2DES. This study considered the mixing of CT and exciton states of Chl, showing three different timescales, 1, ∼6, and 100–200 ps, associated with energy transfer to the CT states in Lhca4.445 Further studies targeted the Lhca1/4 and Lhca2/3 dimers and application of time-resolved fluorescence with streak-camera detection revealed inter-monomer energy transfer occurring on ∼50 ps timescale.446 

Energy transfer processes in the whole PSI-LHCI complex were predominantly studied by time-resolved fluorescence methods, revealing a total trapping time in the 50–150 ps range.447–449 A similar overall trapping time of ∼70 ps was reported also by 2DES applied to intact PSI from pea.450 All four Lhca proteins transfer energy to the core, but the Lhca-to-PSI core energy transfer is faster (∼10 ps) from Lhca1 and Lhca2 than from Lhca3 and Lhca4 (∼40 ps), due to the presence of red Chls in the latter two proteins.451 

c. Carotenoids in energy transfer in PSI

Similar to other light-harvesting complexes, carotenoids also contribute to the energy harvesting and transfer network in PSI. The pioneering study by Kennis et al. employed detection of upconverted Chl fluorescence after excitation of carotenoids in the cyanobacterial PSI core at 510 nm.432 These authors identified two energy transfer channels involving both the S2 and S1 states of β-carotene, characterized by 0.17 and 1.2 ps time constants. Remarkably, the calculated overall carotenoid-Chl energy transfer efficiency was about 90%, thus much higher than for the core antenna proteins CP43 and CP47, which also bind β-carotene. A slightly lower total efficiency was reported in a subsequent transient absorption study on the same system, yet the S2 channel was still found to be very efficient (∼60%). The transient absorption data were, however, better suited to measure β-carotene S1 lifetimes directly, resulting in values in the 3–4 ps range.452 This is shorter than in solution (9 ps) indicating efficient S1 pathway. The dominating and efficient S2 pathway in the PSI core was later confirmed by a low temperature (77 K) fluorescence upconversion study.410 

These studies showed that also carotenoid-Chl energy transfer is highly efficient in the PSI core, with β-carotene S2 lifetimes shortened way below 100 fs due to energy transfer to Chl. It is important to stress that these lifetimes correspond to the average S2 lifetime of the 22 β-carotenes in the PSI core, since, similar to Chls in the PSI core, it is impossible to identify the individual carotenoid-Chl pairs by ultrafast spectroscopy methods. Some heterogeneity in carotenoid-Chl energy transfer was reported for the S1 pathway,452 and it was proposed to be due to the presence of β-carotene cis-isomers in the PSI core, which have higher S1 energy than trans-isomers,187 making the cis-isomers better donors in the S1-mediated energy transfer pathway.

Ultrafast spectroscopy has also been applied to follow carotenoid-Chl energy transfer in LHCI from higher plants. In the first study Gobets et al.433 worked with a preparation containing all Lhca proteins as a mixture of Lhca1/4 and Lhca2/3 dimers. The carotenoids were excited at 470 nm, revealing the presence of only the S2-mediated energy transfer pathway to Chl a, while transfer to Chl b and presence of a fractional S1 route was not reliably identified. Yet, a later transient absorption study focusing only on Lhca4 revealed a minor energy transfer channel from lutein S1 state to Chl a characterized by an ∼3 ps time constant. An efficient S2 channel was confirmed also in Lhca4 and associated with lutein in the L1 site.444 

d. Other antenna systems associated with PSI (FCP-PSI, IsiA)

In addition to the Lhca proteins that expand the light-harvesting capacity of PSI in algae and higher plants, a few other light-harvesting complexes associated with the PSI core have been found in photosynthetic micro-organisms. Diatoms use the fucoxanthin-chlorophyll-protein (FCP) (the energy transfer in isolated FCP is discussed in detail in Sec. III F 2) to enhance the cross-section of the PSI core, forming a PSI-FCP supercomplex, whose structure was determined recently by cryo-EM.453 The whole supercomplex in a diatom Chaetoceros gracilis consists of a monomeric PSI core surrounded by 24 FCP complexes, forming the largest known eukaryotic antenna system described so far. While the energy transfer network in isolated FCP has been studied in detail (Sec. III F 2), much less is known about the energy flow from FCP to the PSI core. Time-resolved fluorescence applied to PSI-only and PSI-FCP from the diatom C. gracilis resolved an extra 20 ps component assigned to energy transfer from FCP to the PSI core,454 and an even faster, 2 ps, component associated with this process was extracted from femtosecond fluorescence upconversion data.455 

A specific arrangement of the PSI antenna was found also in iron-deficient cyanobacteria, which forms a ring, consisting of 18 IsiA protein subunits similar to CP43 (therefore sometimes denoted as CP43′), around a trimeric PSI core.456 Time-resolved spectroscopy revealed energy equilibration processes between IsiA and the PSI core occurring with 2 and 10 ps time constants,457 but only the 2 ps time constant was extracted from transient absorption data a year later.458 It was also shown that isolated IsiA aggregates are heavily quenched, suggesting their photoprotective function under iron deficiency conditions.459,460 Energy transfer from Chl a to the carotenoid echinenone has been suggested as the quenching mechanism.461 A similar ring around the PSI trimer was discovered also in the low-light adapted oxyphotobacterium Prochlorococcus; the ring is composed of 18 subunits denoted as Pcb proteins.462 Transient absorption data showed that the Pcb-PSI complex exhibits an energy transfer network comparable to that observed in the IsiA-PSI complex.463 

What ultrafast spectroscopy taught us about energy transfer and trapping in PSI

  • Energy transfer among Chls within the PSI core occurs on the sub-ps timescale, followed by picosecond equilibration with red Chls in the PSI core, the overall trapping time in the PSI core varies between 15 and 50 ps, depending on species.

  • Outer LHCI complexes occurring exclusively in plant PSI exhibit an energy transfer network comparable to LHCII. The Chl b to Chl a energy transfer occurs on the sub-picosecond timescale, followed by energy equilibration within the Chl a pool on a few picoseconds timescale. The arrangement of LHCI complexes allows for inter-complex energy transfer on a timescale of about 50 ps.

  • Both the PSI core and the outer LHCI antenna exhibit efficient carotenoid-Chl energy transfer that occurs predominantly via the S2 pathway.

  • Other antenna complexes are found to associate with the PSI core, such as FCP, IsiA or Pcb proteins. Energy transfer between these proteins and the PSI core occurs on a picosecond time scale.

1. Light harvesting in peridinin-chlorophyll-protein

Another intensely studied photosynthetic antenna, besides those described in Secs. III D and III E, is the water-soluble peridinin-chlorophyll-protein (PCP) from dinoflagellates. The high-resolution structure of PCP was resolved in 1996 and showed a peculiar pigment composition.464 It is the only known antenna protein having carotenoids as main light-harvesting pigments. PCP forms trimers and each PCP monomer binds 8 molecules of the keto-carotenoid peridinin and 2 Chl a molecules.464 

The first attempts to disentangle excited state dynamics and light-harvesting efficiency of PCP was carried out by Akimoto et al.465 who reported dramatic shortening of the peridinin S1 lifetime from >100 ps in solution to <3 ps in PCP, indicating very efficient energy transfer from peridinins to Chl a. This result was further detailed by measurements of peridinin excited state lifetimes in both solution and PCP.466 Interestingly, however, these new measurements revealed a completely different S1 lifetime of peridinin solution, 13 ps, differing by one order of magnitude from the value reported earlier.465 A subsequent study of the peridinin S1 lifetime in different solvents established a strong dependence on solvent polarity; 160 ps in non-polar hexane and 10 ps in polar acetonitrile.149 The solvent dependence of the S1 lifetime was explained by the existence of an ICT state due to the presence of conjugated keto-oxygen in the peridinin structure. These two reports showed that peridinin-Chl energy transfer in PCP has ∼90% efficiency and that most of the energy goes through the S1-pathway. This is in contrast to other light-harvesting systems containing Chl a, for which the S2 pathway is dominant.11 

Further studies focused especially on the role of the ICT state in energy transfer. Application of global fitting to data measured for PCP over the 450–720 nm spectral region identified two peridinin-Chl energy transfer channels; the sub-100 fs S2 channel accounting for 25%–50% of the total energy transfer, and the S1 channel characterized by a 2.3 ps time constant.467 The involvement of the peridinin ICT state in energy transfer was resolved later, when the spectral range of experiments was extended to the near-IR region where the ICT state is readily identified via its stimulated emission.150 The ICT stimulated emission decay matching the Chl a rise kinetics provided evidence for the ICT state being directly involved in energy transfer.468 Yet, both S1 and ICT states exhibited identical dynamics, leading to the concept of coupled S1/ICT states playing the role of a donor in peridinin-Chl a energy transfer in PCP. Only much later, pump-dump-probe experiments allowed monitoring the dynamics of the S1 and ICT states in PCP separately, showing that the ICT state is the key energy donor in the S1/ICT-mediated energy transfer channel in PCP.469 Interestingly, in contrast to most light-harvesting complexes, the S1/ICT mediated peridinin-Chl energy transfer is faster at low temperatures.470,471

Alternative energy transfer schemes have been suggested based on some experiments, leaving the identification of peridinin-Chl energy transfer channels in PCP an open question. Analysis of transient grating spectroscopy472 and 2DES473 experiments even suggested that all energy transfer occurs from the peridinin S2 state: whereas the fast (sub-100 fs) channel occurs from the Franck–Condon S2 state, the donor in the slower 2.5 ps channel was suggested to be a conformationally distorted S2 state. Yet, these proposals were challenged by another 2DES experiment, which did not identify any contribution from the hypothetical distorted S2 state. Instead, the standard scheme involving the sub-100 fs S2 channel and ∼2 ps S1/ICT channel was proposed.474 However, in contrast to earlier analyses of transient absorption spectroscopy data,467,468,470,475 direct S2-Qy transfer with ∼70% efficiency was proposed, challenging the assignment of the S1/ICT channel as the dominant one in PCP.474 

The PCP complex is amenable to mutation and reconstitution with different pigments,476 which provided an opportunity to systematically examine the factors controlling energy transfer efficiency. Reconstitution of PCP with various Chls showed that when the Qy band of Chl is located close to the maximum of the peridinin S1/ICT emission, the energy transfer efficiency can be even better than in native PCP.477 It was even possible to obtain PCP complexes having different Chls in each of the two binding sites in the PCP monomer,478 further confirming that not only peridinin-to-Chl energy transfer, but also energy transfer between the two Chls present in the PCP monomer occurs, and proceeds via the Förster mechanism as predicted earlier.479 

2. Light harvesting in fucoxanthin-chlorophyll-protein

FCP from diatoms and brown algae is another light-harvesting antenna that has attracted substantial attention thanks to an unusual pigment composition. It has been known that FCP has high homology with LHCII of green plants, except that it has a different pigment composition as it binds Chl a, Chl c and the carotenoids fucoxanthin, diadinoxanthin and/or diatoxanthin.480 The recent high-resolution structure of FCP from the diatom Phaeodactylum tricornutum showed that FCP bears many structural features of the LHCII complex from plants, including conservation of the key pigment binding sites.481 However, FCP features extra carotenoid binding sites that do not exist in LHCII. This results in a carotenoid:Chl stoichiometric binding ratio close to 1 (i.e., 8:9), realized through seven fucoxanthins and one diadinoxanthin plus seven Chl a and two Chl c molecules, thus markedly different from LHCII that binds four carotenoids and 14 chlorophylls (eight Chl a and six Chl b).

The first study targeting the energy transfer pathways in FCP482 demonstrated very efficient energy transfer between fucoxanthin and Chl a that occurred both via the fucoxanthin S2 state and the coupled S1/ICT state of fucoxanthin. The S1/ICT state in FCP has a lifetime of 2.3 ps, which is significantly shorter than the 30–60 ps in various solvents,218,219 suggesting an ∼90% energy transfer efficiency of this channel. The S2 state of fucoxanthin transfers energy with a sub-100 fs time constant.482 Similar fucoxanthin-chlorophyll energy transfer was later confirmed also for FCP trimers and oligomers.483 These authors showed that the particular oligomeric state does not affect the energy transfer channels, but application of different excitation wavelengths suggested a slightly less efficient S1/ICT channel for fucoxanthins absorbing at the red edge of the inhomogeneously broadened FCP carotenoid absorption band.483 

These studies were carried out with FCPs from diatoms, but a very similar picture was reported also for FCP from brown algae, again confirming the dominant (∼70% of total energy transfer) and very efficient S1/ICT channel characterized by an ∼2.5 ps time constant.484 Interestingly, in this FCP the observed dependence on excitation wavelength was opposite to that reported earlier for FCP from the diatom Cyclotella meneghiniana:483 the lowest efficiency of the S1/ICT channel was found for excitation tuned to the blue part of the FCP carotenoid absorption band.484 The same conclusion was obtained from the two-color 2DES data on FCP from C. meneghiniana.485 The latest addition to the knowledge of fucoxanthin-Chl energy transfer in FCP was provided by pump-dump-probe spectroscopy, which enabled addressing the coupled S1 and ICT states of fucoxanthin separately. The data suggest that the S1 part of the S1/ICT potential surface is the major energy donor in the efficient energy transfer to Chl a.486 

To complete the picture of the network of energy transfer in FCP, Chl c to Chl a energy transfer was also studied. The initial study addressed predominantly fucoxanthin-Chl a energy transfer, but global fitting analysis involved also Chl c, concluding there must exist an ultrafast, ∼100 fs, energy transfer channel from Chl c to Chl a.482 A comparable picture was drawn based on fitting data measured on FCP from brown algae: a 100% efficient, ∼100 fs Chl c to Chl a energy transfer.484 The first study that specifically addressed the Chl c to Chl a energy transfer channel in FCP appeared in 2013. A combination of transient absorption and 2DES spectroscopy identified the Chl c to Chl a pathway and found a 60 fs time constant, confirming the highly efficient inter-chlorophyll energy transfer in FCP.487 Later, 2DES carried out at 77 K found two distinct Chl c to Chl a routes operating on different timescales, 320 fs and 3.9 ps.488 The fast one was suggested to be vibronically enhanced while the slower, previously unidentified, channel was proposed to work in the standard Förster regime. All these studies were carried out prior to knowledge of the FCP structure, which indeed resolved two Chl c molecules in FCP. However, the structure was reported for FCP from P. tricornutum, while the key studies addressing the Chl c to Chl a transfer were carried out on FCP from C. meneghiniana. A recent theoretical study showed that the ultrafast spectroscopic data indeed could not be fully explained by the reported FCP structure, indicating that some local differences in FCP structures from different organisms are important for tuning the Chl c to Chl a energy transfer rates.489 

3. Light harvesting in other complexes from micro-organisms

A number of other light-harvesting complexes from various photosynthetic micro-organisms have been studied by ultrafast spectroscopy aiming to disentangle the energy transfer network and to reveal various light-harvesting strategies developed by these organisms. High-resolution structures of these complexes remain unknown, but most of them retain high homology with LHCII or FCP whose structures are available. One such antenna is the Chl a/peridinin/Chl c protein complex (apcPC) from dinoflagellates. It is a membrane complex that accepts energy from PCP and transfers it further to the PSII. The energy transfer pathways in this complex have been a subject of a few studies targeting both Chl c to Chl a and peridinin-chlorophyll energy transfer. The first report showed that peridinin in this complex transfers energy to Chl a predominantly via the S1/ICT state with an ∼2.5 ps time constant, though some fraction is transferred from the S2 state. The Chl c to Chl a energy transfer occurs with a time constant of 1.4 ps.490,491 Other studies focused on spectroscopic properties of the acpPC complex at 77 K,492,493 confirming the overall scheme of energy transfer pathways, though the individual energy transfer rates were slower at 77 K. All studies detected the accessory carotenoid diadinoxanthin, also bound to acpPC, yet its role in the energy transfer network is most likely marginal. Remarkably, despite the completely different structures and pigment composition of apcPC and PCP, the dynamics of energy transfer pathways involving the carotenoid peridinin are very similar in these two complexes. Another antenna that binds a keto-carotenoid is the siphonaxanthin-chlorophyll protein (SCP), found, e.g., in the green alga Codium fragile. It binds Chl a, Chl b, and siphonaxanthin. The siphonaxanthin in this complex transfers energy to Chl a with an efficiency close to unity, most likely via the S1 pathway.494 

Another light-harvesting complex that has been a subject of ultrafast spectroscopy studies is violaxanthin chlorophyll protein (VCP) from Nannochloropsis, a genus of algae that has gained interest due to its possible utilization in biotechnology, because of their high growth rate and accumulation of lipids. VCP binds only Chl a and two carotenoids, violaxanthin and vaucheriaxanthin.495,496 Two spectroscopically distinct pools of carotenoids have been identified in VCP, one with absorption maximum around 480 nm and transferring energy nearly exclusively via the S2 pathway, the other absorbing at 515 nm with a dominant energy transfer pathway via the S1 route.496 Since violaxanthin and vaucheriaxanthin are spectroscopically identical in solution, it was not possible to identify which of these carotenoids contribute to the respective carotenoid pools in VCP. Nevertheless, VCP represents the only known antenna binding a non-carbonyl carotenoid and exhibiting highly efficient carotenoid-Chl a energy transfer via the S1 route. A similar energy transfer network was reported also for VCP from Trachydiscus that has a nearly identical pigment composition but features red-shifted Chl a bands in the absorption spectrum.497 

We list here a few other light-harvesting complexes that have been studied by ultrafast spectroscopy. Liguori et al.498 showed that a stress-related light-harvesting complex LHCSR3 from the green alga Chlamydomonas reinhardtii features an energy transfer network comparable to that of LHCII. There is an efficient Chl b to Chl a energy transfer characterized by two channels, 130 fs and 2.5 ps as well as energy transfer between carotenoids and chlorophylls occurring from both S1 and S2 carotenoid excited states.498 An efficient carotenoid-Chl energy transfer has been reported also for the xanthonema light-harvesting (XLH) complex from the chromophyte Xanthonema, which binds two carotenoids, heteroxanthin and diadinoxanthin, and Chl a.499 Efficient carotenoid-Chl energy transfer was also identified in the Chromera light-harvesting (CLH) antenna complex from Chromera velia. This antenna is closely related to FCP, but binds only Chl a, violaxanthin and a yet unidentified keto-carotenoid,500 which transfers energy to Chl a with an efficiency exceeding 90%.501 Recently, ultrafast spectroscopy data were reported also for a membrane-bound Chl c/Chl a antenna complex (CAC) of cryptophytes,502 which is the only known antenna containing Chl a and Chl c in a 1:1 ratio. Moreover, it binds the carotenoid alloxanthin, the only natural carotenoid with two triple bonds in its structure. A slow, 8 ps, Chl c to Chl a energy transfer pathway was identified in CAC, in addition to the fast channel (∼140 fs) occurring in other Chl c containing antennas. Alloxanthin transfers energy to Chl a, but only via the S2 pathway, while the S1 route is negligible.502 Finally, we mention a unique antenna system, xanthorhodopsin (XR) from Salinibacter ruber, in which a single carotenoid salinixanthin serves as antenna pigment to rhodopsin, enhancing the light-harvesting capacity of XR. Energy transfer between salinixanthin and rhodopsin takes place exclusively from the S2 state with a time constant of ∼170 fs resulting in 40% energy transfer efficiency.503–505 

What ultrafast spectroscopy taught us about energy transfer in other light-harvesting complexes

  • Ultrafast spectroscopy revealed the specific light-harvesting strategy utilizing keto-carotenoids, which employs an ICT state to enhance energy transfer efficiency.

  • PCP utilizes the keto-carotenoid peridinin to harvest light, and the primary energy transfer route is via the coupled S1/ICT state, which takes place on the timescale of a few picoseconds.

  • The same strategy occurs in the FCP complex where the light-harvesting keto-carotenoid is fucoxanthin. In addition to efficient fucoxanthin-Chl energy transfer, efficient Chl c to Chl a energy transfer underlines the robust light-harvesting strategy.

  • In other light-harvesting complexes from various photosynthetic micro-organisms, ultrafast spectroscopy revealed the diversity of light-harvesting strategies developed by these organisms.

The photosynthetic light harvesting summarized in Secs. III B–III F is undoubtedly a key process enabling efficient conversion of sunlight into chemical energy, and ultrafast spectroscopy is an essential tool to disentangle complicated networks of energy transfer channels. In addition to light harvesting, however, there is another light-dependent process required for fitness and survival of photosynthetic organisms. Since nearly all photosynthetic organisms must cope with variations in light intensity, some regulatory mechanisms, ensuring that the photosynthetic machinery will work at a broad range of light intensities, are needed. The central problem is that chlorophylls have quite significant (30%–60%) triplet yield114,115 so if the energy absorbed by singlet states is not exploited for photochemistry, long-lived chlorophyll triplets will accumulate. These triplets may interact with oxygen, resulting in a highly reactive singlet oxygen species, which may damage the photosynthetic apparatus.114 

To prevent such oxidative damage, photosynthetic systems have developed sophisticated photoprotective mechanisms that, under excess light conditions, are able to quench either chlorophyll singlet excited states to prevent triplet formation,506 or chlorophyll triplets if they are already formed,507 or, if the first two protective walls fail, to scavenge singlet oxygen.508 Carotenoids, whose complex network of excited states is described in Sec. III A 2, are the major players in this three-stage photoprotective system. The last two photoprotective stages (triplet quenching and singlet oxygen scavenging) work at nano- to microsecond timescales. The quenching of chlorophyll singlet states, usually denoted as non-photochemical quenching (NPQ), however, occurs at timescales accessible only to ultrafast spectroscopy, which has been commonly used to reveal details of NPQ. It is worth noticing here that in order for photoprotection mechanisms that involve quenching of singlet states to be effective, at least some energy transfer rates in the light-harvesting network of the photosynthetic unit should not be extremely fast, as not to outcompete the photoprotection processes once they are switched on. Thus, there has to be a balance between the said requirement and highly efficient energy transfer, which generally requires moderately fast rates. As has been described in Secs. III B–III F, such energy transfer rates are readily found in photosynthetic units. These rates are typically associated with connection between different light-harvesting systems, as well as being responsible for the final energy transfer step to the reaction center.

Prior knowledge that the xanthophyll cycle, which is an enzymatic light-dependent interconversion of two carotenoids, zeaxanthin and violaxanthin, is important for photoregulation in plants, led in 1994 to the first application of ultrafast spectroscopy to address the mechanism of quenching of chlorophyll singlet states.509 These authors did not attempt to detect the quenching itself, but instead they used femtosecond transient absorption to measure the S1 lifetimes of the involved carotenoids in solution. Then, by application of the energy gap law, they calculated the S1 energies of violaxanthin and zeaxanthin, and concluded that while the S1 energy of violaxanthin is higher than the Qy energy of Chl a in LHCII, for zeaxanthin the S1 energy drops below the energy of the Chl a Qy transition. On the basis of this observation, they proposed the “molecular gear shift mechanism” relying on energy transfer between carotenoids and Chl a: in low light conditions the violaxanthin S1 state serves as energy donor to Chl a, while under excess light violaxanthin is converted to zeaxanthin whose S1 state is low enough to accept energy from the excited Chl a.509 The result is that Chl a excited states would be efficiently quenched due to the short, few ps, lifetime of the zeaxanthin S1 state.

This seminal paper509 proposed the energy transfer mechanism of NPQ and set the stage for further studies addressing this proposal. Later, the S1 energies of violaxanthin and zeaxanthin were measured either by detecting extremely weak S1 fluorescence510 or by the S1–S2 transient absorption.185 These studies showed that although the molecular gear shift mechanism may work, the picture is more complicated, because the difference between S1 energies of violaxanthin and zeaxanthin in solution is much smaller (∼350 cm−1) than the ∼1000 cm−1 prediction based on the energy gap law.509 Further, the development of quantum chemical methods allowing calculation of molecular configurations (defined predominantly by the bond length alternation) of carotenoid excited states, showed that the configurations of carotenoids differ significantly in the S0, S1, and S2 states.511 This means that the energies corresponding to the vertical 0–0 transitions, obtained from spectroscopy experiments, are not necessarily relevant to quenching. Because the molecular configuration in the S1 state is significantly different than in the S0 and S2 states, the minimum of the S1 potential surface is shifted with respect to the minima of the S0 and S2 surfaces (Fig. 17). The shift between potential minima of the S0 and S1 states, denoted Δ in the figure, indicates that the relaxed configuration of the molecule in the S1 state does not correspond to the relaxed configuration in the S0 state, which is consistent with experimental observations.510 The difference in molecular configurations of S0 and S1 states has a significant consequence pertaining to the energy transfer quenching mechanism. A carotenoid in its S0 state able to accept energy from the excited Chl a is in its ground state configuration, implying it has to accept sufficient energy to promote it to a point on the S1 potential surface that corresponds to the molecular configuration of the ground state. This energy, denoted as Ev in Fig. 17, is clearly larger than the 0–0 energy (E0–0) obtained from experiments. Experimental values for this extra energy, ΔE2 in Fig. 17, are not available, but calculations suggest that it is in the range of 1600–1700 cm−1.511 

These observations suggested that to make the energy transfer quenching work, some additional regulation must be provided by the carotenoid binding site in the protein. To measure the carotenoid S1 energies in LHCII turned out to be difficult,512 but single molecule spectroscopy data later identified spectrally distinct quenched conformations in bulk LHCII, or related proteins.513,514 It was thus obvious that to demonstrate the energy transfer mechanism in action, spectroscopic evidence must be obtained for carotenoids in proteins. The first attempt to experimentally detect the carotenoid S1 state after Chl excitation, thereby providing evidence for energy transfer from Chl to the carotenoid S1, was reported in 2003 on thylakoid membranes.515 These authors detected a signal attributable to the carotenoid S1 state, but more convincing evidence was provided a few years later by transient absorption spectroscopy on LHCII trimers for which, after excitation of Chl, the characteristic carotenoid S1 signal was reported.516 Since the energy transfer quenching rate was much slower than the lifetime of the acceptor, the carotenoid S1 state, the S1 signal was not directly visible in the data, but had to be recovered via application of sophisticated fitting methods. Interestingly, the carotenoid lutein bound to LHCII was identified as quencher.516 

A comparable quenching scheme was later reported for IsiA proteins from cyanobacteria where the carotenoid echinenone was suggested to be the quencher.461 Finally, the ultimate experimental evidence for energy transfer quenching was provided by experiments on high-light inducible proteins (Hlip) from cyanobacteria. These proteins are evolutionary related to LHCII, but they are locked in a quenched state. Both transient absorption517 and femtosecond Raman spectroscopy518 unequivocally showed that in Hlips excited Chl a transfers energy to the S1 state of the carotenoid (here β-carotene) within ∼2 ps. The quenching remains equally efficient even at cryogenic temperatures,519 underlining the quenched nature of Hlips. The same quenching mechanism was recently identified also for light-harvesting-like proteins from plants, LIL3 and ELIP.520 An even faster, ∼300 fs energy transfer quenching channel, was detected in 2DES data on LHCII in a more native environment, in membrane nanodisks. Interestingly, however, this quenching channel was observed only for a very narrow range of excitation and probing wavelengths, implying that it probably exists only for unique conformations (energies) of donor and acceptor.521 This again points to the importance of local structural changes for quenching, as suggested also by molecular dynamics simulations on LHCII.522 

A slightly different variant of energy transfer quenching was recently reported for LHCII from mutant plants that synthesize only the carotenoid astaxanthin,523 and in the CP29 minor antenna protein.214 In these two systems, the excited Chl a is apparently quenched via energy transfer, but to the yet-enigmatic S* state (see Sec. III A 2 b). In both cases the quenching channel is slower than the lifetime of the S* state, implying that the quencher characteristics must be extracted from the data by global fitting methods. Involvement of the S* state in the quenching was further reported for LHCII embedded in polyacrylamide gels to prevent aggregation upon quenching induction.524 Here, however, the S* signal is clearly visible in the raw data and decays with the same lifetime as Chl a, but the exact quenching mechanism remains unidentified. Since this mode of quenching appears only when LHCII is embedded in a gel, some local distortions induced by the gel matrix are likely behind this phenomenon.

Although there is experimental evidence that the energy transfer quenching is functional at least in some systems, another quenching mechanism based on ET between carotenoid and Chl was identified by ultrafast spectroscopy. This mechanism, usually denoted as reductive quenching, relies on the ability of carotenoids to give away an electron, thereby reducing a nearby molecule.508 If the excited Chl is reduced by ET from a carotenoid, the generated radical pair then recombines and energy of the excited Chl is harmlessly dissipated. Moreover, the generated carotenoid radical cation is readily detectable due to its strong absorption bands in the near-IR region.525 Quantum chemical calculations predicted that this type of quenching might indeed occur for some geometrical arrangements,526 which was recently corroborated by a combination of molecular dynamics simulations and multiscale quantum chemical calculations applied to LHCII.527 

Experimentally, ultrafast ET between carotenoid and BChl a, following carotenoid excitation was first reported in LH2 from purple bacteria.335 Some fraction (<10%) of excited carotenoids transferred an electron within 300 fs, forming a radical pair with B800 BChl a, which then recombined in ∼10 ps. A possible function of such charge separation in LH2 remains unknown. In 2005, however, Holt et al.528 reported ultrafast transient absorption data on quenched and unquenched thylakoid membranes, which identified a carotenoid (presumably zeaxanthin) radical cation signal after Chl excitation. The Zea+–Chl radical pair formed on the subpicosecond timescale and recombined with a 150 ps lifetime. The key argument for assignment of the radical pair formation to a quenching mechanism was the correlation between the carotenoid radical signal amplitude and the quenching state of the studied sample.528 Interestingly, when searching for a more precise location of this quenching mechanism, no evidence for carotenoid radical formation was found in LHCII trimers, while minor antenna complexes binding zeaxanthin exhibited the carotenoid radical signal in the NIR.529 A more detailed description of the reductive quenching mechanism was provided for the CP29 minor complex, where modeling of the dynamics again yielded a subpicosecond radical pair formation followed by recombination with a 260 ps time constant.530 More recently, a more sophisticated method of measurements, called snapshot transient absorption spectroscopy, again confirmed carotenoid radical formation in thylakoid membranes.531 This method measures a standard transient absorption signal at a fixed time delay for a data collection time of tens of seconds. This allows to obtain the transient absorption signal after application of actinic light, which induces quenching, providing better control of the quenching state of the measured system.531 

Thus, there exists experimental evidence for both energy transfer and ET (reductive) quenching mechanisms. Recent studies suggest that, especially in complex systems such as thylakoid membranes, both mechanisms can be active depending on specific conditions. Snapshot transient absorption spectroscopy demonstrated that the ET mechanism appears within less than 30 s after exposure to high light, while signatures of the carotenoid S1 state signaling the energy transfer mechanism appears later (minutes) after quenching induction.532,533 Both mechanisms were also identified in the LHCSR1 complex from moss Physcomitrella patens. Here, the electron transfer quenching, taking place on a timescale of ∼40 ps was related to pH-induced quenching, while the faster (<10 ps) energy transfer quenching channel occurred only when zeaxanthin was present in the complex.534 Essentially, the same results were obtained for the LHCSR3 protein from C. reinhardtii.535 

A third quenching mechanism, in which the carotenoids are the quenchers, involves formation of a carotenoid-Chl excitonic pair whose lifetime is much shorter than the excited state lifetime of Chl itself. The mechanism was suggested on the basis of quantification of carotenoid S1–Chl Qy interaction by means of a comparison of Chl fluorescence intensity measured either after direct one-photon excitation of Chl, or after two-photon excitation of the carotenoid.536 A clear correlation between the carotenoid-Chl interaction measured in this way and the quenching state of the studied system was demonstrated for both LHCII and whole plants. The same method was later used to provide evidence for this quenching mechanism also in minor antenna complexes537 and even in synthetic carotenoid-tetrapyrrole dyads.538 It is important to note that the increased carotenoid S1–Chl Qy interaction is also important for the previous two mechanisms, energy transfer and electron transfer quenching, because in both cases the switch between unquenched and quenched state cannot be achieved without increased interaction. In fact, a carotenoid-Chl excitonic pair was even suggested as a precursor to formation of the carotenoid-Chl radical pair in CP29.529 It should be also mentioned that recent reports show that in contrast to the original assumption,536 two-photon excitation using a wavelength range corresponding to half of the expected energy of the carotenoid S1 state (1150–1300 nm) does not exclusively excite carotenoids. Instead, a substantial two-photon absorption cross-section has been also found for chlorophylls in this wavelength range.406–408,539

While all three quenching mechanisms described in previous paragraphs involve carotenoids, there is still another mechanism, in which the proposed quenching is not achieved through direct participation of carotenoids. Instead, it is related to formation of Chl–Chl CT states whose presence is manifested by a red shifted fluorescence occurring typically in oligomeric (aggregated) LHCII.540 Ultrafast transient absorption spectroscopy revealed lifetimes of these Chl-Chl CT states in the 5–20 and 200–400 ps ranges.541 This mechanism is likely the cause of the aggregation-induced quenching reported for a number of photosynthetic antenna proteins, but it cannot be the sole NPQ mechanism since quenching can be achieved even without presence of the red-shifted fluorescence in non-aggregated systems.524 

The primary experimental evidence for various quenching mechanisms has been provided by ultrafast spectroscopy and appeared in a relatively short time period between 2005 and 2009.516,528,536,540 The large amount of data collected on this topic during the past decade suggests that there is likely not one quenching mechanism operating under in vivo conditions, but all the mechanisms described above are functional under different conditions and/or different systems. Dall'Osto and co-workers presented the first experimental evidence that at least two NPQ mechanisms are active in higher plants.542 It must be also mentioned that annihilation, which is often present in transient absorption data when measuring on complex systems holding a large number of pigments, such as thylakoid membranes,533 can complicate the interpretation of transient absorption data, and in some cases can even lead to artifactual signals unrelated to quenching. In a report targeting the role of carotenoids in NPQ, van Oort recently showed that experimental artifacts resulting from annihilation could be incorrectly interpreted as population of the carotenoid S1 state via energy transfer quenching.78 

To complete the section on quenching mechanisms, we should also discuss the orange-carotenoid protein (OCP), the key protein related to photoprotection in cyanobacteria. This protein, whose x-ray structure was determined in 2003,543 regulates energy transfer from phycobilisomes to PSII. OCP binds a single carotenoid, in most OCPs reported so far it is either hydroxyechinenone, echinone, or canthaxanthin, making it a much easier object for spectroscopic studies than light-harvesting proteins binding a few different carotenoids and chlorophylls. OCP is photoactive and upon absorption of light converts to a new form characterized by a red-shifted absorption spectrum544 and large rearrangement of the carotenoid position inside the protein.545 The activated form of the protein, denoted as OCPR, binds to the phycobilisome, eventually leading to quenching of allophycoyanin excited states in the phycobilisome core.546,547 OCPR is converted back to the inactive form (OCPO) in the dark with the help of the fluorescence recovery protein (FRP).548 

The first spectroscopic characterization of OCP provided information about excited state properties of the carotenoid. It demonstrated that the S1 lifetime of hydroxyechinenone in OCP (3.5 ps) is significantly shorter than 6 ps measured in solution. The shortening of the excited state lifetime is related to twisting of the terminal ring of hydroxyechinenone to its s-trans configuration, resulting in a large effective conjugation length.549 A similar lifetime shortening was later reported also for OCPR,550,551 confirming that one of the terminal rings remains in the s-trans configuration even in the active form.545 All carotenoids found in OCP are keto-carotenoids, and the keto-group plays a crucial role in forming hydrogen bonds with tyrosine and tryptophan in the binding site.

There is a general agreement that breaking these hydrogen bonds is the first step of the OCP photocycle.552–554 Yet, the very short carotenoid S1 lifetime and rather inefficient (<1%) photoconversion, which makes the detection of the first photoproduct extremely difficult in a time-resolved experiment,544 suggest that the photocycle is not initiated by a reaction involving the excited S1 state. It has been also hypothesized that the ICT state of keto-carotenoids, which has been detected in OCP, may be important for the photocycle, or even for the quenching mechanism.550,555 However, recent data rather suggests that the ICT state is just a side product induced by the binding site without direct relevance to the OCP function.551 Only recently, a combination of UV-VIS and mid-IR transient absorption spectroscopy identified the carotenoid S* state, whose identity is still a matter of debate (Sec. III A 2), as the key player in breaking the hydrogen bonds and initializing the photocycle.215 The same conclusion, the S* state as the starter of the OCP photocycle, was reached on the basis of measurements of tryptophan fluorescence after excitation by 150 fs laser pulses.556 The S*-mediated breaking of the hydrogen bond occurs with an ∼20 ps lifetime.215,556 The low yield of S* in the OCP, which is here assigned to a structurally distorted S1 state, is the reason for the low efficiency of photoconversion.215 

What ultrafast spectroscopy taught us about non-photochemical quenching

  • Ultrafast spectroscopy provided important information about lifetimes and energies of the potential quencher, the carotenoid S1 state.

  • Three different mechanisms involving carotenoids; energy transfer quenching, ET quenching, and a coupled carotenoid-Chl state as quencher, have been identified by ultrafast spectroscopy methods.

  • Another type of quenching via formation of Chl-Chl charge transfer states, identified through red-shifted fluorescence, has been proposed.

  • In cyanobacteria, OCP is the key quencher regulating energy transfer from phycobilisomes to reaction centers. Spectroscopic properties of bound carotenoid (echinenone or canthaxanthin) have been explored. The key role of the S* state in initialization of the OCP photocycle has been identified.

In 1991, coherence signals, observed as oscillations in pump-probe experiments, were reported in the RCs from purple bacteria.81 Soon after, similar oscillations were observed in the LH1 and LH2 light-harvesting complexes from the same bacterium82 and in the chlorosome antenna from green non-sulfur bacteria.557 In both cases, the observed beating signals, dephasing on a picosecond timescale, were interpreted as stemming from superpositions of vibrational states, i.e., vibrational coherences, which were excited by the short and broadband laser pulses. The potential role of these signals for electron and energy transfer was discussed. A few years later, in 1997 Savikhin and co-workers reported coherence signals, observed in the FMO complex at 19 K.279 By analyzing pump-probe anisotropy data the authors could distinguish that they detected electronic (or excitonic) coherences, which were reported to completely dephase in 280 fs. Similarly, a year earlier, analysis of the anisotropy signals in the B820 subunit of LH1 from purple bacteria suggested electronic coherences with 40 fs dephasing time at room temperature.558 All these findings confirmed the understanding that in a “soft” biological system with a huge number of degrees of freedom, any electronic dephasing has to be extremely fast. A decade later, and with the advent of 2DES spectroscopy, FMO was investigated again and the data obtained at 77 K were interpreted as electronic coherences dephasing on up to a ps timescale.83 At the same time, two-color photon echo experiments were claimed to have detected electronic coherences in bacterial reaction centers.559 In both cases, signals were construed as pertaining to energy transfer different from the well-established by that time exciton hopping model. Instead, it was proposed that the signals reported on coherent (quantum like), or wave-like energy transfer. It was also speculated that the protein environment provided protection to this mode of transfer, and therefore slowed down electronic dephasing.559 Later, similar signals were observed in cryogenic 2DES experiments in other light-harvesting complexes560,561 and even at room temperature,562 all interpreted as electronic coherences persisting up to a ps. In principle, such long-lasting coherences could be relevant to the energy transfer timescale (see the discussion on photosynthetic energy transfer above), which led to speculations that nature has designed photosynthetic systems to exploit “non-trivial” quantum effects to optimize photosynthetic functions. This was the basis for the most recent wave of quantum biology to come into a full swing.

Since then, as the experiments, data analysis, as well as theory matured, it has been clearly shown that the said long-lived coherence signals originate not from superpositions of electronic or excitonic states, but are rather vibrational coherences either in the ground or excited electronic states. This has been since shown for bacterial reaction centers73 and FMO,74,563 as well as other photosynthetic systems.488,564 It has been determined that at least some coherences are excited via vibronically mixed transitions,73,74 as has been proposed by theoretical work on non-adiabatic coupling in model photosynthetic systems.565,566 Purely electronic coherences have been also observed, however, even at cryogenic temperatures they have been shown to dephase on the 100-fs timescale, in full agreement with the original coherence paper on the FMO complex.279 This thus clearly demonstrates that electronic coherence cannot play an important role for light-harvesting (for a detailed review see Ref. 84).

Relating any observed coherence signals to photosynthetic processes occurring under incoherent sunlight absorption was discussed in Sec. II C. Recent theoretical work converges to the conclusion that any coherences observed in these experiments are induced by the laser pulses and are not directly relevant to the processes observed in nature.85,567

More generally, when considering the problem of so-called non-trivial quantum effects in photosynthetic light harvesting, one can look at the issue through the lens of the measurement problem, which is notoriously tied up with different interpretations of quantum mechanics. The information which can be obtained is inseparably entangled with the type of measurement that is carried out on the system. The relevant inquiry is then what kind of “measurement” a photosynthetic system (or in fact any light-driven system) in a living organism “performs” when it utilizes absorbed sunlight. It is reasonable to assume that in this physiological measurement quantum effects cannot be traced, because in a sense they are averaged over.

Then, an important question becomes clear—are studies of coherence signals useful for understanding of the mechanisms found in photosynthetic systems, or of molecular photophysical functions in general? The answer is yes, since the studies of coherences can be and are used as a tool to learn about the properties of molecular systems, which are important for the photophysical and photochemical functions. Polarization-controlled 2DES experiments used to investigate coherences excited by vibronically coupled transitions constitute one group of examples. These experiments enable investigation of subtle molecular properties, such as vibronic mixing that is beyond the Condon as well as Born–Oppenheimer approximations. Such studies have shown that vibronic mixing between electronic or excitonic states is ubiquitous, and it has been identified in chlorophyll molecules72 and various photosynthetic complexes.73,74,568 Another example of the usefulness of coherence signals as a tool pertains to employing them to identify the nature of molecular transitions, observed in absorption. This was demonstrated in a study of porphyrin nanorings, where either electronic or vibronic origin was assigned to all transitions in the near IR absorption region.569 

Light-harvesting—some unresolved issues

During the past three decades, ultrafast spectroscopy has provided enormous amounts of information about energy transfer pathways, mechanisms, and strategies of light-harvesting utilized by antenna proteins of various photosynthetic organisms. Although it may seem that the key questions have been answered and the knowledge provided by ultrafast spectroscopy allows us to understand in detail the light-harvesting strategies of all main groups of photosynthetic organisms, there are still a lot of questions awaiting answers. First, most of the spectroscopic studies targeting the light-harvesting pathways and/or mechanisms have been carried out with isolated antenna proteins in a buffer containing detergent to prevent aggregation of antenna proteins outside their natural environment, the photosynthetic membrane. While this approach is no doubt important to reveal the basic picture of the energy transfer network, recent studies have indicated that this non-natural environment may slightly alter the structure of the complexes, thereby affecting the spectroscopic properties and interactions between pigments.521,570 Thus, designing ultrafast experiments with antenna complexes in their natural environment is a challenge for the near future.

Similarly, the light-harvesting systems of essentially all photosynthetic organisms consist of multiple antenna proteins, yet the detailed knowledge about energy transfer between these proteins, such as, e.g., LH2 and LH1 in purple bacteria, PCP and acpPC in dinoflagellates, or LHCII and minor antenna proteins in PSII, remains elusive. Accordingly, the ultrafast studies of whole photosynthetic units containing a complete light-harvesting system are desired. Here 2DES is a promising spectroscopic tool as it is capable of disentangling contributions from pigments that spectrally overlap in traditional transient absorption methods. This capability of 2DES was demonstrated in monitoring the energy flow through the complete antenna system of green sulfur bacteria.284 To this end, recent advances in cryo-electron microscopy leading to emergence of a number of high-resolution structures of photosynthetic systems represent an important step forward, because the structural knowledge is the key guide to understanding of spectroscopic data taken on the whole photosynthetic unit.

Furthermore, new methods of ultrafast spectroscopy, such as 2D electronic-vibrational spectroscopy,65 femtosecond stimulated Raman spectroscopy (Sec. II B 3 a), or combining ultrafast spectroscopy with single-molecule spectroscopy,571,572 hold promise to open new horizons in ultrafast spectroscopy of light-harvesting systems. These new tools can provide so far unknown information about dynamics of pigment–protein interactions at a molecular level (e.g., FSRS), or to reveal the importance of conformational switching in tuning the light-harvesting capacity (ultrafast SMS). Last but not least, the slowly emerging spectroscopic approaches utilizing quantum light (Sec. II C) will hopefully enable the study of energy transfer processes with light having properties corresponding to physiological conditions which photosynthetic organisms experience in nature.

The RCs found in all photosynthetic organisms are the place where the excitation energy is converted to an electrochemical potential. This energy is later used in photosynthetic dark reactions to synthesize energy-rich compounds such as sugars. The understanding of the functions of RCs was facilitated by determination of the first RC structure (from purple bacteria) by Michel and co-workers in 1984.573 As more RC structures were revealed through the years from different photosynthetic organisms,354,415,574 it was realized that whereas there is a wide variety of light-harvesting complexes (see Secs. III B–III F) in different photosynthetic organisms, featuring varying architectures, which help these organisms to optimize light capture in their environment, the RC structure in all photosynthetic organisms is largely preserved. The main chromophores participating in the chain of ET are six chlorophyllides arranged in two symmetric or pseudo-symmetric structures. Two of these chlorophyllides form a strongly interacting dimer, called a spatial pair (P), which serves as the electron donor.

There are two types of RCs distinguished, depending on the main electron acceptors: type I, where iron–sulfur clusters serve as acceptors, and type II, featuring quinones as acceptors. It is understood that the charge separation processes in RCs have to be fast—at least in the range of 10 ps. This is required, because energy collection and transfer from light-harvesting complexes to the RCs happens on the timescale of 20–100 ps (see Secs. III B–III F), and, with the help of the charge separation event, the energy has to be trapped during this time window to prevent efficient back energy transfer from happening. This is of high importance, because the special pair in RCs is usually surrounded by many antenna pigments and therefore back energy transfer from the special pair can be very efficient. During the ET event, charges are usually separated by a 20–30 Å distance in the time window of ∼200 ps. This large separation distance also assures inefficient charge recombination. Additionally, during the CT process a substantial amount of energy is dissipated (substantially more than the thermal energy), which results in losses of power conversion efficiency (PCE), but energetically further ensures prevention of charge recombination.575 

It is worth noting that compared to the energy transfer processes, where the distance between the donor and acceptor, as well as resonance conditions matter the most, the ET depends on more parameters. In addition to the distance, redox potentials of the different states of the cofactors and thermodynamic parameters play an important role. These properties seem to be finely tuned by the protein environment surrounding the chlorophyllide cofactors. This has been demonstrated in multiple mutation studies,576–580 where change of even one amino acid was shown to have a very large effect on ET, whereas energy transfer usually remains unchanged. Perhaps because of the requirement to have a delicately tuned structure, nature managed to “come up” with only one robustly working design (so far) which is reused in all organisms.

As mentioned above, the structure of RCs always comprises two branches, which have high mutual symmetry. This fact is related to one of the biggest mysteries regarding the function of reaction centers—it has been proven that in some photosynthetic organisms the ET happens along both branches, and in many others, ET preferentially or exclusively proceeds through one of the branches.

1. Electron transfer in reaction centers from purple bacteria

Through the years, ultrafast spectroscopy played a crucial role in understanding of the ET processes in photosynthetic RCs. Such studies using 10 ps lasers were pioneered by Kaufmann et al.,7 Rockley et al.,98 and Shuvalov et al.581 Large excess energies, as well as huge intensities were used in these experiments, and time resolution to resolve all the ET steps was lacking. Nevertheless, it was shown that primary ET in RCs takes place on the several ps timescale and that an electron arrives to the non-chlorophyllide electron acceptor in hundreds of ps. All these studies concerned type II RCs in photosynthetic purple bacteria. The crystallographic structures have revealed that these RCs comprise a dimer of BChl a molecules, called special pair (P), two accessory BChls a (BA and BB), two bacteriopheophytin (Bpheo) molecules (HA and HB) as well as two quinones (QA and QB) (see purple bacteria RC structure in Fig. 18). In the absorption spectrum the P, B and H bands are observed around 760, 800, and 870 nm, respectively, but they do not strictly correspond to the absorption of individual molecules, but rather to excitons with some delocalization over two or more chromophores.576,582 Already in the eighties, it was demonstrated that ET occurs with up to 99% selectivity via the active branch on the A side forming the P+QA ion-radical pair.583–585 A closer look at the structures revealed that the B branch has slightly larger intermolecular distances and that the two branches feature different amino acids close to the chromophores. This asymmetry was concluded to cause preferential ET through the A side.576 

As laser pulses became shorter and ultrafast spectroscopy techniques kept improving, experiments with moderate excitation fluences and 100 fs resolution became available. This led to the finding that the ET from P to HA takes place on the timescale of 2.8 ps at physiological temperatures.586,587 The presence of accessory BChls between the special pair and Bpheos has been known from the structures, so it came as a surprise that an electron seemed to be transferred directly from P to H. This was interpreted with the help of the superexchange mechanism, where BA was understood to play a role of mediator increasing the coupling between P and HA. From this moment the issue of direct or indirect participation of BA in ET became hotly debated. The issue was seemingly settled by Zinth and co-workers, who used a series of different probing wavelengths, including the spectral areas where BA could be directly detected.588 They convincingly showed that ET proceeds first from P to BA, then to HA and finally to QA with the time constants shown in the scheme below:

PhvP*2.8psP+BA0.9psP+HA200psP+QA.

The authors explained the complication of detecting the BA signal as follows; because it is populated slower than it is depopulated the transient amplitude of BA is always small, impeeding any spectroscopic detection.

Even though the general ET scheme in RCs from purple bacteria has been established, there are still many details that have not been understood. Van Grondelle and co-workers suggested the presence of an alternative CT pathway, where the BA* serves as an initial electron donor and HA as an acceptor;589 however, this proposal is very difficult to reconcile with the fact that BA* transfers energy to the special pair on the timescale of 100 fs,582 which would outcompete the suggested potential ET process. A closer look at the evolution of transient spectra of all three majors bands in the near infrared region (P, B, and H) revealed a high complexity, and proved to be very challenging to interpret.590,591 One of the issues is a very strong transient electrochromic B band shift signal resulting from the electric field between P+ and H, which is established after the initial ET.591–593 Even the nature, number, and position of electronically excited states have not been completely determined. For example, the energy assignment of the higher exciton state of the special pair (P+), which was traditionally thought to be located at 825 nm at room temperature,594 has been challenged in recent 2DES spectroscopy studies,568,592 which proposed a location at 840 nm at 77 K.

It has been shown with the help of Stark spectroscopy that the state corresponding to the P band has strong permanent dipole moment, indicating involvement of CT states;595,596 however, the energetic location of the pure CT states and how they mix with the purely excitonic P states have since been debated.597,598 Even the apparent double structure of the P band as seen in low-temperature absorption measurement of some bacterial RCs599,600 still waits for a consistent explanation.

Another direction of studies on RCs concerned coherences observed in time-resolved spectroscopy experiments.71,81,559 Recently, it has been clarified that direct involvement of coherence in RC function is questionable, especially of electronic coherences.84 It has not even been established if coherences can be excited in principle by the absorption of photons coming from the Sun, which could be considered as a thermal source,85,567 or if it is just an “effect” of RCs being excited with broadband ultrashort laser pulses (for more details, see Sec. II C).

2. Electron transfer in reaction centers from oxygenic organisms

Primary charge separation in higher organisms occurs in two photosynthetic units, photosystem I (PS I) and photosystem II (PSII), and the RCs found in these photosystems belong to the type I and type II, respectively. Both PS work in a concerted way to oxidize water and transfer electrons to NADP+, creating ATP molecules in the process. In contrast to bacterial RCs discussed in Sec. III I 1, the absorption spectra of the main cofactors in the PSI and PSII RCs overlap with the absorption of the chromophores in the light-harvesting antennas that surround them. This is a big challenge for spectroscopic studies of ET, since it cannot be easily disentangled from the energy transfer in these systems.

a. Electron transfer in photosystem II

The PSII monomer consists of at least 19 protein subunits, but fortunately the RC subunit (RC D1/D2) can be extracted and detailed spectroscopic studies can be carried out on these isolated units. The RC D1/D2 unit features 6 chlorophyll molecules arranged in the two branches A and B in the D1 and D2 proteins, respectively. Two closely positioned Chl a chromophores form a special pair absorbing at 680 nm (P680).354 There are also two accessory chlorophylls a, ChlD1, and ChlD2, two pheophytins (PheD1 and PheoD2) and two plastoquinones (QA and QB). Thus, the PSII RC structure is very similar to the structure of bacterial RCs. In addition, in the PSII RC there is a bicarbonate ion bound to a non-heme iron between QA and QB, which serves as a bridge for electron and proton transfer between plastoquinones.

Despite the spectral congestion, dynamics of charge separation in PSII have been a subject of highly intense studies through the years, starting with pioneering work by Shuvalov and co-workers,601 which showed that an electron is transferred from the excited special pair P680* to Pheo and then to QA. As in the bacterial RCs, ET in PSII was also shown to proceed only along one, so-called active branch (D1).602 There have been many other studies of ET transfer in PSII (see, for example, Refs. 603–605) which provided information on the rates and processes involved. However, there is still no widely agreed upon consensus on the precise processes and the role of different chromophores.575 What seems to be agreed upon is that there are more than one possible primary electron donor and acceptor pairs and at least two different charge separation pathways. Historically, the first pathway which was determined included PD1 and PheoD1 as primary electron donor and acceptor, respectively, and ET was concluded to proceed in the following way:606 

(P680ChlD1)*0.9psP680+ChlD114psP680+PheoD1250psP680+QA.

In several further studies, the alternative ET pathway was reported and characterized.605,607 It was found that the initial ET occurs from ChlD1 to PheoD1, thus forming the ChlD1+PheoD1 radical pair on a sub-picosecond times scale, followed by ET from P860 to ChlD1+, and energy transfer from PheoD1 to QA. Both charge separation pathways were found to substantially contribute to the photochemical reactions in PS II.

Furthermore, with the help of 2DES, coherence signals in PS II RCs were measured and analyzed.608,609 It was determined that the observed coherences are vibronic in origin, which indicated the important role vibronic transitions play in efficient charge separation in the RC of PS II.

b. Electron transfer in photosystem I

The pigment–protein complex PS I is found in both prokaryotic (cyanobacteria) and eukaryotic (algae and higher plants) photosynthetic organisms, and the same cofactors are found in the ET chain of PS I. The fact that the RC core of PSI cannot be extracted from the native PSI complex without losing its function implies that any functional preparation of PS I has ∼100 chlorophylls per monomer, all absorbing in the similar spectral range. This is a severe complication for studies of ET in PS I.

The ET chain of the RC in PS I, which is type I, consists of a Chl a dimer special pair P700 (Chl1A/Chl1B), two pairs of Chl a molecules designated as Chl2A/Chl3A and Chl2B/Chl3B, together regarded as electron acceptor A0, and the iron-sulfur clusters FX, FA, and FB.415 In addition, PS I also includes two molecules of phylloquinone (A1A and A1B) in between the A0 cofactors and FX. In contrast to bacterial RCs and the PS II RC, the ET in PS I occurs through both branches from P700 to FX.610 An interesting structural detail of the ET cofactors is that the planes of the intermediate chromophores Chl2A and Chl3A as well as Chl2B and Chl3B are oriented parallel to each other and have a very short distance between their centers of ∼8.5 Å. This orientation and proximity cause excitonic interaction, leading to the appearance of the excitonic states delocalized over these chromophores.

Because of the high spectral congestion of the system and interference with energy transfer signals in PSI, the kinetics of primary charge separation and the nature of primary electron donor and acceptor are still debated.611 The first picosecond experiments on PS I612,613 led to the suggestion that the initial electron separation took place on the timescale of ∼14 ps, when P700+A0 ion-radical pair was formed. Since then, multiple time-resolved studies put the primary charge separation step from P700* to A0 in the 0.8–4 ps time range and the following ET from the primary electron acceptor A0 to A1 in the 10–50 ps range.274,614 Subsequent ET from A1 to FX was determined to take place on the times scale of 20–200 ns.610,615 However, there has been a report proposing ultrafast initial charge separation on the order of 100 fs between P700 and A0.611 To elucidate the reason for the big discrepancies in ET times found in the literature, Semenov et al.616 did a systematic time-resolved study including tuning of the excitation wavelength. The result was that different experimental conditions resulted in different interpretation of data on the initial states and dynamics in PS I. The authors came to the conclusion that it is nearly impossible to isolate ET signals from antenna energy transfer dynamics. Thus, a clearer assignment of ET dynamics in PSI waits for further systematic experiments, perhaps the ones with high temporal and excitation frequency resolution (such a 2DES at low temperature) and/or involving mutants of PS I, where contribution of antenna dynamics is dramatically reduced.

From available data, it appears that primary ET in both PS I and PS II are similar: a strongly interacting Chl dimer serves as the primary electron donor with ET to a primary electron acceptor in the time range of 10–30 ps, while the secondary ET takes place by reducing quinones. Whereas this step in PS I has a characteristic lifetime in the range of tens of ps, in PS II it is ∼250 ps, thus an order of magnitude slower. If these ET times in fact correspond to the actual situation, they can be explained by the difference in distance between A0 and A1 in PS I, and PheD1 and QA in PS II, the latter being substantially longer.575 

What ultrafast spectroscopy taught us about electron transfer in reaction centers

  • In bacterial RCs, the energies of all electronic states have been identified and step-by-step ET between the electron chain co-factors has been characterized.

  • The very high efficiency of charge separation across the RCs was shown to be assured by the very fast (in the range of 3 ps) initial ET process.

  • Because of the strong spectral congestion in RCs from oxygenic organisms, ultrafast spectroscopy provided a whole range of possible ET scenarios and corresponding rates.

  • The ET in the photosystem II RCs was shown to have more than one primary electron donor and acceptor pair and consequently at least two different charge separation pathways.

  • It has been established that in contrast to all the other RCs mentioned here, both branches of photosystem I RCs are active in ET.

Electron transfer in reaction centers—some unresolved issues

  • Perhaps the biggest remaining question is why some of the RCs have asymmetric ET via one of the branches and how did the different types of RCs with symmetric and asymmetric ET evolve?

  • The related question concerns the molecular mechanism of said asymmetric ET. Is dielectric asymmetry, different redox potentials, or something else responsible for it?

  • To what extent the protein environment is responsible for assuring fast and directional ET across RCs remains to be established. Can fast protein dynamics control this chemical reaction, as has been suggested?

  • Menaquinone or phylloquinone is present in some of the type I RCs. What is its role for ET?

  • The presence and type of pure CT states in the special pair of RCs still has to be clarified. Is there such a state low in energy, mixing efficiently with excitonic states of the special pair to ensure directional charge transfer?

  • Most of the details of ET, such as the nature of the primary electron donor and acceptor and the rates of charge separation, remains to be clarified in photosystem I.

Lessons from studies of energy and electron transfer in photosynthesis

  • In a properly organized system of pigment molecules with an energy gradient of excited states, directed ultrafast energy transfer on tens of ps timescale can be achieved over several tens of nm.

  • Charges can be moved on picoseconds timescale over many nanometers through sequential downhill ET processes.

  • Spontaneously aggregated chromophore systems often lead to efficient quenching of excited states, but with the help of proteins, nature is controlling chromophore distances and orientations such that quenching is avoided and energy or electron transfer optimized.

  • The challenging photophysics and functions of carotenoids were largely resolved by ultrafast studies.

  • Combining ultrafast energy and ET results with high resolution structural data has provided detailed pictures of energy flow through the antenna/RC systems of several photosynthetic organisms.

  • It took nature millions of years to develop photosynthetic machineries to perfection; this may suggest that building artificial systems for solar fuel production by mimicking nature is probably a very challenging task. Nevertheless, photosynthesis is a formidable source of inspiration and studies of its processes have given invaluable insights into the physics and chemistry of light energy conversion.

  • Coherence signals have been universally observed in all photosynthetic complexes, but should be viewed as an unavoidable consequence of the use of ultrashort pulses in the femtosecond experiments and not as something playing a role in photosynthetic function.

Similar to natural photosynthesis discussed above, light-harvesting naturally constitutes the first key step to initiate solar energy conversion in synthetic solar energy conversion systems including both solar electricity and solar fuels (photocatalytic) applications. In molecular devices, this is typically accomplished by designated light-harvesting/chromophore units, often referred to as photosensitizers. A broad range of both organic and inorganic photosensitizers has been developed and optimized for different applications, and new light-harvesting materials continue to be developed for novel and improved applications.

Photosensitizers are generally selected to match the particular application requirements in terms of suitable excited state properties, and typically include strong and spectrally broad light absorption, as well as long-lived photoactive excited states. These choices inevitably include consideration of any ultrafast dynamics such as internal conversion, intersystem crossing and solvent relaxation that cause excited state energy losses relevant for the viability of the excited state to drive subsequent energy conversion reactions.

A wide range of organic chromophores are available for molecular light-harvesting applications. In many cases, the main photophysics is relatively simple and involves the photo-excitation and spontaneous deactivation of the first singlet excited states. The spin-allowed de-excitation back to the ground state often limits the excited state lifetime in widely used chromophores such as perylene derivatives to the lower end of the nanosecond timescale. While the basics of such simple photophysics is well-understood,617 this often makes them useful photosensitizers for applications based on fast ET, such as dye-sensitized solar cells (DSCs) and, together with corresponding polymer analogs, organic photovoltaics as discussed below. It can be noted that in some cases the excited state dynamics becomes more involved, for example, in cases when intersystem crossing is sufficiently efficient to allow significant involvement of the energetically lower analog of the first excited singlet state before decay of the excited state back to the ground state. There is an increasing amount of research aiming to utilize more advanced photophysics in molecular devices, for example, through exploitation of so-called thermally activated delayed fluorescence (TADF).618 Recently, there has been an increase in interest in yet more unconventional photoelectrochemical properties of organic molecules, such as the exploitation of excited organic radicals that, for example, in the case of rylenes, show sufficient stability to warrant interest for device applications.619 

Transition metal complexes play a prominent role as photosensitizers in many solar energy conversion applications including both molecular photovoltaics and homogeneous photocatalytic applications despite often comparatively modest absorption cross sections (∼1000–10 000 M−1 cm−1) compared to many organic chromophores (∼10 000–100 000 M−1 cm−1). The typically quite limited absorption cross sections of the metal complexes are often compensated by longer excited state lifetimes extending to hundreds of nanoseconds or longer in many CT and metal-centered excited states. Another prominent feature of many photoactive systems involving transition metal complexes with versatile oxidation state properties includes the capability to accomplish multi-electron redox processes. This makes transition metal based molecular systems widely employed in many solar energy conversion applications, for example, with diffusion controlled photocatalysis applications.

Due to a combination of wide significance for solar energy conversion and related photochemical applications combined with often more intricate and versatile photophysics, the ultrafast dynamics of transition metal complexes has remained a very active field of research until the present day. Many of the fundamental photophysical and photochemical properties of transition metal complexes have been well documented in many reviews, see, for example, Ref. 620 and references therein. In the following, some of the prototype properties and recent developments relevant specifically for solar energy conversion applications are briefly outlined.

In particular, many ruthenium(II)-complexes have favorable excited state properties for solar cells and produce among the best DSCs.621 They are also considered as catalysts for solar fuel production (see Sec. IV F).

The early photoinduced dynamics in these complexes is generally characterized by an initial metal-to-ligand CT excitation from a singlet ground state (1GS) to low-energy excited singlet (1MLCT) state, i.e., 1GS → 1MLCT. From a molecular orbital perspective this corresponds to promoting an electron from a t2g level on the metal center to a π* level on the ligand. In contrast to many simpler chromophores that typically remain in the lowest excited singlet (S1) state, there are often rich ultrafast dynamics in transition metal complexes that have been explored in many detailed photophysical investigations. In addition to internal vibrational relaxation (IVR) and solvent relaxation dynamics typically taking place on a few ps timescale, this is characteristically followed by internal conversion within a manifold of excited states as well as intersystem crossing to a set of corresponding triplet metal-to-ligand charge transfer (3MLCT) states involving orbitals of the same type as the 1MLCT states. A combination of heavy-element effects on spin–orbit coupling and the relatively flexible metal-ligand bond coordination structure lead to significant state mixing and associated ultrafast femtosecond timescale state-to-state transitions involving both spin allowed and spin forbidden excited state processes.622–624 A prototype example of key deactivation processes in a Ru-polypyridyl complex with a long-lived and photochemically active 3MLCT excited state is illustrated in Fig. 19.

A significant problem with Ru and other second and third row transition metals is that these metals are very rare and sometimes poisonous—clearly not ideal for large scale implementation of the technology. From the perspective of large-scale and low-cost solar energy conversion applications, it has therefore been a long-standing ambition to replace the rare and expensive Pt-group metals with inexpensive and widely available earth-abundant elements such as iron, zinc, or copper instead of ruthenium.627,628

Iron is a particularly obvious choice as an earth abundant, inexpensive, and environmentally benign lighter congener to the very scarce element Ru.629,630 However, its intense MLCT absorption has been considered unexploitable in energy conversion applications, due to the low-lying metal-centered (MC) quintet high-spin state that typically deactivates the 1,3MLCT manifolds on a sub-picosecond timescale.630–635 The typical difference in excited state structure between polypyridyl complexes based on ruthenium and iron is illustrated in Fig. 19.

From Fig. 19, it is clear that destabilizing the MC states provides a strategy to achieve longer lived MLCT states. To this end, an iron N-heterocyclic carbene complex (Fe-NHC) {[Fe(CNC)2] (PF6)2, [CNC = 2,6-bis (3-methylimidazole-1-ylidine)] pyridine} was recently synthesized, and ultrafast transient absorption measurements revealed a 100-fold extended 3MLCT state lifetime of 9 ps,636,637 as compared to previously known FeII-polypyridyl complexes (∼100 fs).630,638 Additional work has resulted in other Fe-NHC complexes with further extended excited state lifetimes, modified absorption properties, and improved understanding of their photophysical properties.639–642 Quantum chemistry density-functional theory (DFT) and time-dependent density-functional theory (TD-DFT) calculations637,643,644 showed that the exceptionally long excited state lifetime of Fe-NHCs is achieved through a significant destabilization of both triplet and quintet metal centered states compared to other FeII-complexes, mainly as a result of strong σ-donation from the NHC-ligands.637,642 A shallow 3MLCT potential energy surface with an indication of a low activation energy along the transition path from the 3MLCT to 3MC, and facile crossing from the 3MC state to the ground state, were identified as additional key features for the remarkably long lived 3MLCT state (relative to traditional Fe-complexes), very short lived 3MC and the fact that the 5MC state is never populated (Fig. 20).

Chabera et al. demonstrated photoluminescence (PL) in a low-spin hexa-NHC Fe(III) complex.645 This complex was found to display fluorescence from an excited state of ligand-to-metal charge-transfer (LMCT) type that has been observed in d5 complexes.646–648 The lifetime of this state was observed to be 100 ps, record long for an iron complex at that time. LMCT excited states are significantly less explored in Ru(II), Fe(II), and other d6 complexes,649 compared to the standard MLCT excited states. From a fundamental photophysics point of view, it is interesting to note that the strong sigma-donation of the NHC ligands appears to strongly influence the excited state dynamics of both MLCT and LMCT states through destabilization of MC states involved in the deactivation. This opens up for further work to explore the ultrafast dynamics and energy conversion capabilities of the d5 complexes featuring the LMCT excitations more broadly. Recently, another Fe(III)-NHC complex with a strongly luminescent 2LMCT excited state having a significantly longer lifetime of 2.0 ns and fluorescence quantum yield of 2% was reported.650 Stable organic light-emitting diodes (OLEDs) based on this complex have been successfully produced, suggesting that Fe(III) complexes exhibiting spin-allowed doublet metal-to-ligand charge-transfer (2MLCT) emission may be the basis for future low-cost OLEDs.651 

On a different track, McCusker and co-workers have explored electrochemical and photophysical properties of the iron complex [FeII(dcpp)2]2+ [dcpp = 2,6-bis(2-carboxypyridyl)pyridine] that utilizes expanded cage ligands forming six-membered metallocycles to approach the 5T2/3T1 crossing through increased ligand field strength associated with a more octahedral metal coordination as a useful strategy to influence the deactivation pathway via metal-centered states.652 Damrauer and co-workers have explored another unconventional strategy to achieve Fe(II) complexes with long-lived photoexcited MLCT states through the use of modified terpyridyls as highly strained ligands to exploit high-spin (quintet/septet) state manifolds,653,654 reaching a 17.4 ps lifetime for [Fe(dbtpy)2]2+ (where dbtpy is a bromine modified terpyridyl).

More recently, still another sensitizer design, in which Fe (II) centers are supported by frameworks containing benzannulated phenanthridine and quinoline heterocycles paired with amido donors [Fe(RL)2 where R = Bu or CF3 added for increased solubility], produced complexes with absorption covering the entire visible spectrum and nanosecond (∼2.6 ns) CT excited state lifetimes.655 A combination of steady state and transient absorption spectroscopy with TD-DFT calculations showed that this was enabled by strong mixing between amido nitrogen and iron orbitals. In order to understand why such orbital mixing should supersede, the impact of the anticipated decrease in ligand field of Fe, an aerobically stable Fe(ClL)2 complex with a 3 ns excited-state lifetime was synthesized.656 X-ray absorption spectroscopy and resonance inelastic x-ray scattering at the Fe L3-edge and N K-edge were used to validate the strong Fe−Namido orbital mixing responsible for the panchromatic absorption and demonstrate a competition between ligand-field strength and metal–ligand (Fe−Namido) covalency that stabilizes the 3CT state over the lowest energy triplet metal-centered (3MC) state, which leads to the nanosecond excited CT lifetime. These findings highlight metal–ligand covalency as a design principle for elongating excited state lifetimes in abundant metal photosensitizers.

The continued development and in-depth characterization of a broad range of Fe(II) and related earth-abundant photosensitizers with improved photophysical properties continues to be a very active area of research as evident from several recent publications,657,658 and also detailed in several recent reviews.10,659–662 This includes recent work on controlling ultrafast dynamics through synthetic manipulation of excited-state vibronic coherences. Vibronic coherences were observed in ultrafast time-resolved absorption measurements of Fe(II)-based chromophores after MLCT excitation. Following visualization of the vibrational modes associated with these coherences, a modified Fe(II) complex was synthesized to interfere with the specific atomic motions. The redesigned compound exhibited a MLCT lifetime more than 20 times longer than that of the parent compound. This suggests that the structural modification at least partially decoupled the vibrational degrees of freedom from the excited state population dynamics. These results indicate that excited state vibronic coherence may be exploited to tailor ultrafast excited-state dynamics through targeted synthetic design.663 

A dye-sensitized solar cell (DSC) consists of a thin film of metal oxide nanoparticles, often TiO2 or ZnO, sensitized to visible light by dye molecules attached to the nanoparticle surface (Fig. 21). Light absorption by the sensitizer dye results in electron injection into the metal oxide nanoparticle followed by diffusive electron transport through the nanoparticle film. The electrical circuit is closed by a liquid or solid electrolyte, which also has the function of restoring the oxidized sensitizer to its neutral ground state before the next photon is absorbed.664–666 Processes of importance for an efficient solar cell are dye-semiconductor electron injection, electron–hole recombination, regeneration of oxidized dye by the electrolyte redox couple, charge transport through the nanostructured film, and finally extraction of electrons into an external circuit.667–669 The timescale of these processes range from femtoseconds to milliseconds and longer; in this review, we will focus on the initial ultrafast injection and recombination processes as well as on the mobility of charges injected into the semiconductor. From a solar cell PCE point of view, these processes are of utmost importance—every photon not resulting in a conduction band electron and every electron–hole pair recombining imply decreased efficiency.670,671 Further summaries of most aspects of DSC function and processes can be found in many reviews and books.672–678 

1. Photons become charges—electron injection from sensitizer to semiconductor

Initial work on excited state and electron dynamics in DSC materials focused on the dye to semiconductor electron injection process. A large body of work identified this process as decisive for efficient light-harvesting and conversion of the light energy to energy-rich electrons.666,668,669,673,679–687 For efficient utilization of absorbed photons and excited state energy of the sensitizer, electron injection into the semiconductor has to be significantly faster than the sum of all other excited state deactivation processes. For many of the sensitizers explored, nanosecond and longer excited state lifetimes are not unusual, implying that injection times on the few ps timescale is sufficient for close to 100% quantum efficiency injection. Nevertheless, from a fundamental point of view, the precise mechanism of dye-to-semiconductor electron injection is interesting and has attracted significant theoretical and experimental attention.

Interfacial ET dynamics in materials for practical DSCs are significantly influenced by structural and energetic heterogeneity, caused by a variable surface binding of the sensitizer. Calculations of ET dynamics in such systems are therefore non-trivial, if at all possible. Calculations have, however, been performed and compared to experimental results on well-controlled systems. For instance, a series of perylenes attached to TiO2 substrates using different anchor and spacer groups by Willig and co-workers provided a good test set for computational validation of ultrafast electron injection models in the so-called wideband limit, i.e., with the excited perylene donor level situated well above the conduction band edge of the TiO2 substrate.688–690 These investigations highlight the significant and non-trivial role of the anchor and bridge groups as mediators of interfacial ET through a chemical influence on the interfacial electronic coupling that overrides simple arguments based on physical distance between chromophore and surface.690–692 Heterogeneous ET was found to take place on a sub-100 fs timescale for a set of several anchor-cum-spacer groups and depending significantly on the particular choice of linker group. Injection with a prototype carboxylic acid anchor group was determined to take place on a ∼10 fs timescale. Remarkably, the injection with an unsaturated (ethene-type) spacer resulted in a very similar fast injection despite the significantly longer through-linker distance, while a saturated (ethane-type) linker of similar length resulted in a significant slow-down of injection to several tens of fs,693 and could be corroborated computationally.690 

Following this short detour to DSC ET calculations in model systems, we continue our account by discussing the ultrafast electron dynamics in DSC materials widely used in “real-world” solar cells. Next, we will summarize some work intended to develop sensitizers based on benign and earth abundant elements and compare the excited state/electron dynamics of these sensitizers with that of the traditionally used ones. Finally, some work on ultrafast dynamics in functional DSCs will be summarized.

RuN3-sensitized nanostructured TiO2 is the active material that for a long time gave the highest efficiency (∼10%) DSC. Figure 22 illustrates the dye-TiO2 attachment in such a DSC electrode. As discussed in more detail above (Sec. IV B), the electronic structure of the Ru-polypyridyl complexes extensively used as sensitizers include singlet and triplet metal-to-ligand CT states (1MLCT and 3MLCT, respectively), accounting for the strong visible absorption. Light absorption by these molecules therefore generates the excited 1MLCT state, but within a very short time (∼100 fs) the molecule relaxes to the lowest 3MLCT state. This implies that for a complete description of the injection process, injection from both the short-lived 1MLCT and 3MLCT states needs to be considered. For efficient electron injection and energy conversion in the sensitized semiconductor system, the energy of the lowest 3MLCT state has to be above the conduction band edge of the semiconductor. The resulting scenario is illustrated in Fig. 23, showing the valence and conduction bands of the semiconductor, as well as the ground and excited states of the RuN3 sensitizer. Following light absorption to the 1MLCT state, ∼50% of the molecules inject electrons directly from this state into the semiconductor conduction band with a characteristic time constant of ∼50 fs.679,681,683,684,694 Upon excitation to higher-lying vibrational states of the 1MLCT state, even faster injection occurs (∼20 fs), in competition with vibrational energy relaxation and redistribution. The residual ∼40% of the excited sensitizers relax to the triplet state, from which they inject electrons much more slowly on the 1–100 ps timescale.695,696 Many other Ru-sensitizers,669,697 but also porphyrins,698,699 phthalocyanines, and organic dyes700–702 have by now demonstrated fast and efficient injection. Thus, to achieve efficient electron injection from a sensitizer dye to a metal oxide nanostructured film appears to be a relatively manageable task for chromophore-semiconductor interfaces that give sufficient driving force for electron injection and interfacial electronic coupling of the excited state with the substrate conduction band.

The overwhelming part of the work addressing the injection process has been performed on electrode systems in contact with a solvent. Work on full solar cells is considerably less common but has certainly been performed and was recently reviewed.672 For example, electron injection dynamics in complete solar cells based on the N719 ruthenium bipyridyl sensitizer were studied by transient absorption and emission experiments and were correlated with charge recombination and device performance.703 It was found that the electron injection dynamics depend on the composition of the redox electrolyte employed in the device. The half time of electron injection from the 3MLCT state at optimum photovoltaic device efficiency was 20 times slower than that for control dye-sensitized films covered in inert organic liquids. This retardation was shown to result from the influence of the electrolyte on the conduction band energetics of the TiO2 electrode. In a later paper by the same authors, it was shown that a 100 mV up-shift of the conduction band edge leads to twofold retardation of injection dynamics.704 It was further concluded that optimum DSC performance is obtained when the charge separation kinetics are just fast enough to compete successfully with the dye excited-state decay, and at the same time minimizing interfacial charge recombination losses.703,704

The ratio between ultrafast singlet and slow triplet injection is an issue addressed in several papers on complete solar cells, with results in qualitative agreement with work on DSC thin films in contact with solvent. Nevertheless, more quantitatively, some papers report mainly ultrafast,705,706 but others mainly slow707,708 injection. The reasons for this discrepancy can probably be found in differences in singlet/triplet injection rates of the two ruthenium polypyridyl sensitizers used, RuN3 (fully protonated) and N719 (partially deprotonated), as well as differences in sensitivity to electrolyte components and other additives in the complete solar cell.

Interfacial electron dynamics involving sensitizers anchored on alternative semiconductor substrates of interest for DSC applications have also been investigated extensively.665,673,675,710 ZnO has in particular, provided a good material for comparison with TiO2 due to its similar bandgap. Calculated interfacial electronic interactions using organic model adsorbates were found to be significant for ZnO as for TiO2 suggesting that ultrafast electron injection could occur for both substrates.711 Experimental evidence for multiexponential electron injection of Ru polypyridyl complexes on ZnO nanocrystalline thin films with an ultrafast (<100 fs) as well as slower (tens to hundreds of ps) components was presented by several groups.712–714 This multiexponential slow injection was shown to be a result of formation of an exciplex (or electron–cation complex) upon sensitizer excitation (see below).675,713,714 Surface ET kinetics have also been investigated for numerous other substrates such as, e.g., SnO2, with opportunities to vary the interfacial electronic interaction, driving force for injection, and surface passivation.715 Electron injection was found to be non-exponential with sub-ps and ps components, similar to injection to TiO2, though with a somewhat higher percentage of the ps timescale injection.

2. Lost charges—electron–cation recombination

Electron injection from dye to semiconductor is just the first step in a series of processes that eventually lead to a PC in an external circuit. For the electrons to be extracted with a high yield, recombination with the holes on the oxidized dye has to be much slower than the re-reduction of the oxidized sensitizer by the redox couple of the hole transport material (HTM). Since this process relies on diffusion of redox components in a liquid electrolyte, or charge migration in a solid HTM, the rate of re-reduction may vary greatly depending on the nature of the HTM.665 Electron–hole recombination times on the hundreds of ns and slower timescale are generally sufficient for efficient utilization of the light generated charges. The realization that electron–hole recombination is a process directly related to solar cell efficiency—every recombined electron is a lost electron and lost PC—has motivated work to understand the factors controlling the process.672,673,703 For Ru-polypyridyl dyes (e.g., RuN3, N719, and the black dye) resulting in very efficient solar cells, electron–cation recombination has been shown to be very slow (microsecond timescale)716,717 and slower than regeneration of the oxidized sensitizer by the redox mediators,718 and therefore generally not a limiting factor for the efficiency of a solar cell based on these dyes. Charge recombination kinetics in such dye-sensitized films have, however, been found to be strongly dependent upon the electron occupation in trap/conduction band states of the TiO2 film (up to a million-fold change in rate). This occupation may be modulated by variations in light intensity, applied electrical potential, and electrolyte composition.719 Slow recombination for some champion Ru-polypyridyl sensitizers under optimized conditions often seems to have been extrapolated to suggest that this is also the case for other dyes, leading to a picture where variations in solar cell efficiency have been directly correlated with the efficiency and rate of electron injection (see, e.g., Ref. 720). Electron–cation recombination kinetics is generally observed to be strongly material (sensitizer and metal oxide) dependent and highly non-exponential, covering a very broad timescale from picoseconds to milliseconds.675,721,722 The strong non-exponentiality of the process is generally explained as a consequence of trapping–detrapping from trap states with a wide distribution of energies.721,723,724 However, the strong dependence on sensitizer of the recombination rate as well as theoretical considerations,690,725 suggest that there are also factors related to, e.g., sensitizer structure, exact mode of binding to the metal oxide surface, etc., controlling the recombination dynamics.

The free energy driving force of the interfacial injection and recombination is an important parameter, and it should be minimized for injection and large for recombination for efficient DSCs. These parameters have been studied, but with significant dispersion of the results that indicate that other factors also may be significant for efficient performance.726,727 The mode of sensitizer binding to the semiconductor surface can be anticipated to be important for the interfacial ET. In order to provide further insight into this issue, a systematic study was performed of how sensitizer binding to the semiconductor surface controls the ET processes in general, and electron–cation recombination in particular.698 By using a series of Zn-porphyrins (Zn-P) attached to nanostructured TiO2 films in contact with solvent, several molecular properties of importance for dye-semiconductor binding could be varied systematically.669,698,699 The excited state of the sensitizer was formed within the time resolution (<100 fs) of the experiment and was converted with multi-exponential kinetics to the oxidized sensitizer, which then decayed back to the ground state by charge recombination with conduction band electrons. This recombination could for most dyes be described by two lifetimes, one on the tens to hundreds of picoseconds timescale and another much slower, >50 ns.

The distance dependence of charge injection and recombination was examined with the help of porphyrins having a variable length spacer to the semiconductor surface (Fig. 24). If ET between the porphyrin core and the semiconductor occurs via the connecting spacer, as often envisaged, making this spacer longer should, as predicted by Marcus theory of ET,728–732 slow down the transfer. Transient absorption kinetics of the molecules attached to the TiO2 film showed that charge recombination does not meet this expectation—the sensitizer with the longer connecting spacer had a much faster recombination rate than the molecule with the shorter spacer.698 Also, the electron injection was faster for the longer spacer. The acceleration of ET was even more pronounced for an analog to the sensitizer with short spacer having a less bulky porphyrin core, with very fast injection and complete recombination within 500 ps698 (Fig. 25). Obviously, for these porphyrin sensitizers, ET does not occur as could be anticipated via the spacer connecting the porphyrin core to the TiO2 surface. Instead, a picture was suggested where the single carboxyl anchoring group allows a flexible binding geometry; for some of the porphyrins, depending on structural factors such as length of the spacer group and bulkiness of the porphyrin core, a fraction of the molecules are bound at an angle to the semiconductor surface and ET occurs through space rather than through the linker group connecting the porphyrin core to the anchoring COOH group (Fig. 24).698 When the tilt angle is changed as a result of a change of porphyrin molecule size or shape, the distance between the porphyrin core and semiconductor surface changes, which will lead to a change in the through-space ET rate. Owing to the expected exponential distance dependence of ET, only a modest change of distance (and thus angle) will have a dramatic impact on the transfer rate.

In order to further establish this picture of the porphyrin sensitizer binding to TiO2 vibrational sum frequency generation spectroscopy (SFG) was used on Zn porphyrins labeled with a CN infrared active chromophore.699 The IR transition dipole moment of the CN-group is along the symmetry axis of the Zn-porphyrin molecules; SFG therefore gives the orientation of the porphyrin relative to the semiconductor surface (the tilt angle). These measurements showed that there is a direct correlation between tilt angle of the Zn porphyrin molecule and amplitude of long lived (>50 ns) conduction band electrons that can contribute to PC—smaller tilt angle leads to higher amplitude of long-lived electrons. By comparing tilt angles obtained from the SFG measurements with solar cell power conversion efficiency, this correlation was taken one step further—a smaller tilt angle leads to higher cell efficiency, meaning that solar cell power conversion efficiency is directly related to the extent of slow electron–cation recombination. Several groups have in this context explored advanced anchor strategies, and studied the electron transfer dynamics, with the aim to achieve better functional control of the sensitizer–semiconductor interactions and to achieve more effective interfacial CT properties that may be relevant not only for traditional dye-sensitized solar cells but also for related applications with, e.g., photocatalysis on sensitized semiconductor films.733–739 

3. Useful charges—formation of mobile charges

In order to extract photogenerated electrons from the active material they have to be mobile—in the first place to move them away from the place they were created and avoid recombination with the holes, and second to transport them to the interface where they are extracted from the active material. As we have seen, optical spectroscopy can provide detailed information about excited states and intermediates participating in the light-to-charge conversion process of DSC materials, but optical techniques unfortunately do not provide much information about charge mobility and transport. More information is obtained from transient far-infrared conductivity spectra and kinetics measured by TRTS.48,714 This technique allows non-contact characterization of photoconductivity with sub-ps time resolution,740,741 where the amplitude of the measured photoconductivity signal is a direct measure of the population of injected charge carriers and carrier mobility. In addition, from the shape of the transient conductivity spectrum it is possible to infer the mechanisms of the charge transport or to distinguish between the response of free charge carriers and localized excitations.46,742 The strong interaction of THz radiation with free charge carriers in semiconductors makes the TRTS an ideal tool for the investigation of charge carrier dynamics in semiconductors and DSCs. Time-resolved microwave conductivity (TRMC) provides similar information, but probes a different part of the mobility spectrum.743,744

The conventional scenario for DSC is that mobile electrons appear in concert with injection. Optical transient absorption studies675,713 had suggested that there is a delay in formation of free charges for a dye-ZnO system. A combined optical TA/TRTS study was used to simultaneously monitor the electron injection through formation of oxidized sensitizer (by TA) and appearance of mobile charges (by TRTS) for two different dyes, a Zn-porphyrin and RuN3, attached to ZnO or TiO2.46,714 For both dyes on TiO2 mobile electrons appeared within the time resolution of the experiment (<1 ps) and decayed very slowly. This agrees well with the ultrafast formation of oxidized sensitizers observed in many studies (see above and Refs. 679 and 698). When the sensitizers were attached to ZnO the picture was dramatically different. For both dyes, mobile electrons appeared very slowly on the several hundred ps timescale, whereas oxidized dye appeared on the ∼1 ps timescale. The difference in electron dynamics for sensitized TiO2 and ZnO was rationalized by the reaction scheme in Fig. 26. For dye/TiO2 formation of mobile charges is direct and ultrafast, whereas for ZnO an electron–cation complex714 (or exciplex673,675) is first formed. This complex can then either dissociate into a free electron and cation or recombine back to the ground state. The reason for this difference in formation of mobile charge carriers in ZnO and TiO2 is the difference in screening of the electrostatic interaction in the two metal oxides, due to the much higher dielectric permittivity of TiO2—80 vs 8 for ZnO.714 The fact that this difference in electron–cation interaction for sensitizers attached to TiO2 and ZnO results in fast recombination in a ZnO electrode would adversely affect the efficiency of a solar cell based on ZnO. Using the same combination of ultrafast techniques slow, several hundred ps, dissociation of “CT states” was also observed at N719/TiO2 interfaces in contact with I/I3 redox couple components.745 The dissociation time was seen to depend on the presence of small ions (Li+) and excitation wavelength—shorter with Li+ and at shorter excitation wavelength providing excess energy to the dissociation process.

4. Earth abundant metal-based sensitizers

For large-scale implementation of DSCs, sensitizers based on rare and expensive transition metals (e.g., Ru and other second and third row transition metals) are not a viable choice; sensitizers must consist of abundant, cheap, and nontoxic elements. In order to examine the potential as sensitizer in a DSC, a FeNHC complex with carboxyl anchoring groups at the electron-accommodating pyridine moieties was synthesized and attached to a nanostructured TiO2 electrode.746 Electron injection from the photoexcited Fe-complex was characterized using a combination of electron paramagnetic resonance, optical TA and time-resolved THz spectroscopy. Taken together, the results of these measurements showed that electron injection occurs at high yield. The THz kinetics provided quantitative characterization of the electron dynamics showing that injection occurs with a 3 ps time constant at 93% yield (Fig. 27). Electron–cation recombination is highly non-exponential proceeding on the timescale tens of ps to many ns. The slow ns recombination only accounted for ∼15% of the overall concentration of charge separated states, explaining the low efficiency of early solar cells based on this complex.747 More recently, several groups have achieved efficiencies of Fe-NHC-based DSCs around 1%. Becker and co-workers reported an optimized efficiency of ∼0.9%,748 while Lindh and co-workers obtained a 1.3% efficiency by introducing a linearly aligned push-pull geometry of the Fe-ligands.749 Ultrafast and efficient electron injection was observed, but similarly ultrafast recombination reduced the number of electrons contributing to photocurrent to only 10%–15% of the injected population. For this sensitizer-TiO2 combination, minimizing electron–cation recombination appears to be the obvious way to increase power conversion efficiency. The highest efficiency so far for a Fe-NHC-based DSC was reached for a series of six Fe(II)NHC-carboxylic sensitizers, with the ligand not bound to TiO2 decorated with functions of varied electronic properties—a maximum conversion efficiency of 1.83% for the best sensitizer, and exceeding 1% for all six sensitizers.750 

Transition metal complexes based on Cr(0), Mo(0), and W(0) have recently also been investigated as “d6 analogs” of Fe(II) and Ru(II) complexes.751 Copper complexes, in particular bis-diimine Cu(I) type complexes, have also received considerable interest as potentially inexpensive and earth-abundant photosensitizers for DSCs.752 Anchored organic chromophores can also be used for efficient electron injection to TiO2. Another way to achieve visible light sensitization that has been extensively explored is by semiconductor quantum dots (QDs). The ultrafast electron dynamics involving these sensitizers share the basic features with the sensitizers discussed above. For details, we refer to the recent review by Ponseca and co-workers.625 

What ultrafast spectroscopy taught us about dye sensitized solar cells

Energy and electron dynamics have been studied in a multitude of dye sensitized nanostructured semiconductor materials. Transition metal complexes, porphyrins, and organic dyes as sensitizers to metal oxides like TiO2 and ZnO have been investigated. Innovative ligand design for Fe-complexes has triggered hope that such complexes will be useful in energy conversion applications. From an impressive body of work, a detailed picture of the light induced processes can be painted.

  • Electron injection from photoexcited sensitizer to semiconductor acceptor often occurs on the ultrafast, fs to ps, timescale, but cases of much slower injection are also known. The injection time depends on factors like attachment geometry and electronic structure of the components. The ratio between injection time and sensitizer excited state lifetime in the absence of injection controls the injection efficiency. Thus, for a sensitizer with nanosecond or longer excited state lifetime, injection on the ps time scale is sufficient for close to 100% efficiency.

  • For efficient electron injection, it is of course necessary that the lowest excited state of the sensitizer is located above the conduction band edge of the semiconductor acceptor. Transition metal complexes, with photoactive singlet and triplet MLCT states, are a bit special in that respect. Light absorption by the 1MLCT state is followed by ultrafast electron injection, in competition with intersystem crossing to the lower lying 3MLCT state. For maximal injection efficiency, it is necessary that the 3MLCT state is situated above the conduction band edge of the semiconductor.

  • Historically, the efficiency of a DSC has often been directly correlated to electron injection rate and efficiency. More recent studies have shown that recombination between the injected electron and positive hole on the oxidized sensitizer is at least as, if not more, important for DSC efficiency. The rate of recombination can vary over a wide range, depending on properties of the sensitizer. For the best solar cells, recombination is in the microsecond, or slower time range, while in unfavorable cases it can even be in the picoseconds. Sensitizer attachment geometry (and rigidity) has been shown to be important in this respect—a flexible attachment allowing close contact between the chromophore part of the sensitizer and the semiconductor can result in a kind of “short circuit” of the charge separated state and ultrafast back-ET (i.e., recombination) from semiconductor to oxidized sensitizer.

  • For PC generation, extraction of the electrons from the nanostructured semiconductor has to be efficient, which requires mobile electrons. It has been shown that the electrostatic interaction between the injected electron and the positive charge on the oxidized sensitizer can lead to an electron–cation complex (or exciplex), which limits the electron mobility by confinement to the vicinity of the positive hole at the semiconductor surface. The electron–cation complex also constitutes a gateway to charge recombination. Screening of the charges in a high dielectric constant semiconductor (i.e., TiO2) and moving the positive hole away from the semiconductor–sensitizer interface by chemical design of the sensitizer are the means to reduce/eliminate this unwanted effect.

  • For a long time, Ru-complexes as sensitizers produced the best solar cells. Ru is, however, a rare and expensive metal making large scale implementation of DSCs based on such sensitizers unlikely. DSC sensitizers (as well as other components) have to be based on more abundant and benign components. Through optimization of redox couples and other electrolyte components, the best DSCs are now produced with Zn-porphyrin sensitizers. Recent research work on metal complexes of Fe and Cu, previously considered of very limited interest for energy conversion applications, has sparked hope that these earth abundant and benign metals will become useful in sensitizers and catalysts. As an example, by novel ligand design Fe-complexes with photochemically active CT states of nanosecond lifetimes, instead of the traditional femtoseconds, have been developed. DSCs with FeNHC sensitizers have been developed and have reached a power conversion efficiency of ∼1%.

Dye sensitized solar cells—some unresolved issues

  • New sensitizers based on abundant elements like Fe and Cu are now appearing. The first step toward exploring their usefulness in a solar cell context is to characterize basic photophysics and photochemistry, including processes on the fs to ns timescale.

  • Next step in the search for new DSC sensitizers is to investigate the light driven processes, electron injection, separation and recombination, as well as carrier mobility, all requiring the information ultrafast spectroscopy can provide.

  • An, often far from intuitive, correlation between sensitizer structure, binding geometry and rate of electron injection and recombination has been established (see above). With the appearance of new sensitizers there is generally only one way to establish this correlation—measure the electron injection and recombination processes with appropriate time resolution, i.e., sub-ps.

  • It has been shown that the conditions in a solar cell, i.e., electrolyte and other additives, may substantially alter the energetics in the cell and therefore the ET processes. More work in operando on full devices is certainly necessary.

Organic solar cells offering the possibility of facile solution processing and a wide range of material designs is an interesting possibility to achieve low-cost photovoltaic devices, now with a reported PCE more than ∼18% (https://www.nrel.gov/pv/cell-efficiency.html). Using relatively simple solution chemistry protocols, light-harvesting and acceptor molecules are mixed together in order to prepare the active material. This is in contrast with silicon-based solar cells that require high temperature processing as well as a clean room for fabrication. The electron and hole in Frenkel excitons formed by light absorption by the light-harvesting molecules have high binding energies, on the order of several hundreds meV, much higher than the thermal energy at room temperature (25 meV). This is in contrast with the very low (∼10 meV) binding energy of Wannier–Mott excitons in inorganic semiconductors. In order to break a molecular exciton and form free charges ET to an electron accepting material is therefore required. The most successful strategy to achieve this in organic solar cells is with the BHJ concept—light-harvesting (donor) and electron-accepting molecules are blended on the nanometer scale. This results in nanometer-sized domains of neat donor and acceptor materials and a large interfacial area. Excitons in the organic donor materials have low diffusion rate and length of approximately 10 nm, hence the need for small donor domains to achieve high yield of charge separation at the donor/acceptor interface. Large interfacial area provides extensive donor/acceptor contacts for charge separation. For over two decades, the most studied organic bulk heterojunction material is the polymer/fullerene system, where the polymer absorbs light and bound electron–hole pairs (excitons) are generated in the polymer. Fullerenes, whose conduction band is lower than that of the polymer, then accepts the photoexcited electron and transports it to the electrode. The generally accepted picture is that electrons are transferred to the fullerene balls on the ultrafast, sub-picosecond, timescale leaving free holes on the polymer chains, which are eventually extracted at the counter electrode.753–755 This is illustrated in the schematic shown in Fig. 28.

Over a period of more than 20 years, the PCE of polymer:fullerene solar cells slowly increased from a few percent to ∼12% (https://www.nrel.gov/pv/cell-efficiency.html), while on par with DSC, not quite competitive with other established solar cell technologies. Solar cells using fullerenes as electron acceptors in addition suffer from stability problems, difficulties to fine-tune the electronic properties as well as high costs. Around 2011 non-fullerene electron acceptors (NFAs) started to appear, based both on small molecules and polymers.756–762Figure 29 illustrates the development of small molecule NFAs and the remarkable progress in solar cell PCE based on NFAs. In 2017 PCE of NFA solar cells took the lead over the traditional fullerene-based cells, and as of early 2021 has reached a value of ∼18%.763,764

In the following, we will start by discussing energy and charge carrier dynamics of polymer:fullerene BHJ materials and solar cells. This will be followed by a short account of the short history of the corresponding processes in polymer:NFA materials and solar cells.

1. Charge generation in polymer: Fullerene organic solar cells

Charge carrier generation is a critical, and often non-trivial, step on the path to high solar energy conversion efficiencies in OPVs.766,767 One of the earliest reports on the ultrafast charge transfer in these materials showed a distinct feature at about 1 ps in the transient absorption spectra of the poly(3-octylthiophene-2,5-diyl) (P3OT) polymer in the presence of C60.768 This was concluded to be due to ultrafast ET from the polymer to fullerene and that the presence of C60 at a few percent concentration increased the photoconductivity of the polymer by an order of magnitude. The kinetics in Fig. 30 are the results of another transient absorption study employing sub-10-fs excitation pulses to monitor the CT rate.769 Photoinduced kinetics of neat poly[2-methoxy-5–(3′,7′-dimethyloctyloxy)-1,4-phenylenevinylene] (MDMO-PPV) (line) and MDMO-PPV:PCBM (PCBM = [6,6]-phenyl-C61-butyric acid methyl ester) (open square) are shown for the first 600 fs. The ΔT/T kinetics probed at 580 nm exhibits an ultrafast rise for both samples, assigned to stimulated emission. For MDMO-PPV:PCBM the subsequent decay has a time constant of 45 fs, and was concluded to be due to quenching of stimulated emission by the ET process. The much slower decay of the excited neat MDMO-PPV polymer represents the deactivation of the polymer excited state in the absence of an electron acceptor.

A systematic study of charge transfer was performed for a APFO3:PCBM blend {APFO3 = [2,7-(9,9-dioctylfluorene)-alt-5,5-(4′,7′-di-2-thienyl-2′,1′,3′-benzothiadiazole)]} in a series of papers from the Lund/Linköping/Gothenburg collaboration group.770–772 In a first study, quenching of APFO3 excited state absorption on the few-hundred femtosecond timescale in the APFO3:PCBM blend was taken as a sign of ultrafast ET from polymer to PCBM; the excited state decay of the neat polymer was much slower, several hundred ps.770 Ultrafast ET was further supported by the failure to detect any photoluminescence in a streak camera experiment with ∼2 ps instrumental response time. A follow-up study showed that the efficiency of charge generation is highly dependent on the concentration of PCBM in the blends. Already at a PCBM concentration of 20% (PCBM/polymer w/w) the exciton to charge conversion is almost 100% efficient and at blend ratios of 20%–50% optimal charge formation was obtained.771 However, at a low blend ratio of 5% the exciton signal was not quenched, showing that the solar cell performance of this interpenetrating network of polymer:PCBM is not limited by the charge formation under optimal PCBM/polymer ratios, but by energy transport if the PCBM concentration is too low. In a third study, varying of LUMO levels of the acceptor by slightly modifying the acceptor molecule provided a more detailed account of the charge transfer.772 APFO3 blended with the fullerenes [60]PCBM, [70]PCBM, and [70]BTPF exhibited a qualitative difference in the charge generation rate. Transient absorption kinetics showed that the charge generation times with [60]PCBM and [70]PCBM as electron acceptor are similar (0.2 ps), while for [70]BTPF the generation time is approximately half (0.1 ps). This matches the trend in free energy change of ET estimated from the LUMO energies of the polymer and fullerene derivatives.773 

Hwang et al. also investigated the effect of morphology on charge generation774 in blends of regioregular poly(3-hexylthiophene-2,5-diyl) (rrP3HT) and PCBM. They concluded that mobile carriers are generated via a two-step process—an initial ultrafast charge separation to a bound CT state, followed by the transfer of carriers onto the polymer and PCBM networks. Recombination occurs from the bound CT state before it dissociates, reducing the yield of mobile carriers. When annealed, the number of charge carriers lost due to recombination was reduced by a factor of 2.5, consistent with the increase in photovoltaic efficiency.774 

A different approach was taken by Muller et al. using a pump-push technique with PC detection.775 The pump pulse excites the donor polymer and a delayed pulse of sub-bandgap energy results in a change of PC, either due to exciton depletion (leading to PC decrease) or polaron pair excitation and dissociation (leading to PC increase). In this way, the experiment can distinguish between formation of bound polaron pairs and free charges by the first pump pulse. With this method dramatically different charge carrier generation behavior was detected for two different polymer:PCBM blends. When for the MeLPPP:PCBM (MeLPP = methyl-substituted ladder-type polyparaphenylene) blend, the second push pulse was tuned to the wavelength of polaron pair absorption, a distinct increase in PC was observed. It was concluded that for this material, photogenerated excitons mainly dissociate into Coulombically bound geminate polaron pairs with a high probability for recombination, consistent with an observed low PC yield. On the other hand, for the MDMO-PPV:PCBM blend, the push pulse only caused a small decrease in PC due to exciton depletion and it was concluded that ET leads to the formation of free charge carriers with high efficiency, without the need of additional help by the push pulse. It was also concluded that geminate charge recombination significantly reduces PC yield and depends on the very local interfacial polymer:fullerene structure.

More recent work using a similar pump-push-probe approach, but with optical probe,776,777 suggested that delocalized band states formed immediately after exciton dissociation may act as the gateway for charge separation. It was proposed that charge separation in efficient organic photoconversion systems occurs through hot-state charge delocalization rather than energy gradient driven intermolecular hopping. This work was extended to include the dependence of charge separation on driving energy and state availability (through fullerene phase morphology), with the help of a series of fullerene derivatives and by varying the polymer:fullerene ratio.778 Using ultrafast transient absorption spectroscopy with sub-20 fs resolution, electron transfer and charge separation was studied in model polymer:fullerene BHJ systems. Charge generation times ranged between 37 and 240 fs, depending on driving energy and blend ratio. For the polymer:fullerene 1:4 (w/w) ratio, times vary between 37 and 75 fs and for the 1:1 ratio between 54 and 240 fs (Fig. 31). Thus, in the blends with higher fullerene ratio the charge generation was observed to be considerably faster, almost one order of magnitude for a polymer:fullerene blend with a particular driving energy. This shows that the charge generation time is controlled by two factors, the driving energy and the fullerene cluster size. As in Ref. 776, the latter aspect was concluded to correspond to the number of available states for ET, illustrated in Fig. 32. In this picture, ultrafast CS occurs in competition with formation of strongly bound CT states (electron–hole pairs), which are the gateway to geminate recombination, a loss process. These results also suggested that ultrafast and efficient charge generation can occur with very small driving energy. To gain more insight into the nature of the bound CT states temperature dependent pump-push-photocurrent probe experiments, similar to those in Ref. 775, were performed.778 A push pulse of 2000 nm was chosen to dissociate the CT state by exciting the bound positive hole-polaron, but avoiding ground state excitation of the polymer. For two 1:1 blends of MDMO-PPV with the PCBMs, mono-PCBM and tris-PCBM, exhibiting a high yield of bound CT states, the push pulse generated a large increase in PC with an activation energy of 86–166 meV, 3–5 times kT at room temperature. This shows that the CT states in these polymer:fullerene blends will not dissociate thermally into free charges. This fact and the short 1 ns lifetime of the CT states of these polymer:PCBM blends explains why they reduce the PC of a solar cell.

Time-resolved photoemission spectroscopy measurements on a model system Zn-phthalocyanine/C60 interface also implied high CT state binding energy.779 It was found that photoinduced charge transfer in 150 fs populates delocalized interfacial CT states with a coherence size of 4 nm, which in 2 ps localizes to smaller (1–3 nm) and lower-energy CT states. For this particular system, the localized CT states have binding energies of 0.2–0.5 eV, explaining their long lifetime (at least 300 ps, limited by the measured time window) and silence in PC generation. In still another pursuit of charge generation, using sub-ps to μs broad-band transient absorption, it was shown that polymer exciton dissociation in a PCDTBT:PCBM blend leads to the formation of free charges and Coulombically bound CT states, in a branching ratio of 89% for free charges and 11% CT states.780 The CT states were found to have a lifetime of 2.5 ns, too short to contribute to PC in a device. Despite the high yield of free charges, the power conversion efficiency of their devices was only 3%, and it was concluded that the energetic disorder in the blend leads to charge trapping in solar cells, which in turn leads to higher carrier concentrations and more significant non-geminate recombination.

In a somewhat different approach charge separation was visualized in a P3HT:PCBM BHJ blend. Using ultrafast time-resolved-electric field-induced-second-harmonic (TREFISH) measurements,781 the charge drift following photogeneration was directly measured.782 It was shown that initially, immediately after photoexcitation, only closely separated (<1 nm) charge pairs are created and that diffusion of the unthermalized charges separates them by several nanometers during the first few picoseconds. By ∼100 ps the carriers have separated to a distance of ≥5 nm, where Coulomb interaction is effectively broken. This time to convert bound charge pairs to free mobile charges is much shorter than typical geminate charge recombination times (∼1–100 ns),783 explaining the efficient interception of geminate recombination and high yield of free charges in an efficient solar cell. Numerical simulations complemented these experimental results and showed that fast three-dimensional charge diffusion within an energetically disordered medium, augmented by the correlated entropy change, is sufficient to drive the charge separation process. The picture of charge generation and separation emerging from this work is illustrated in Fig. 33. A similar picture was observed for a bilayer structure784—dissociation of Frenkel excitons results in charge pairs of less than 4 nm separation distance, which under the influence of the electric field escape the Coulomb potential and split into free charges. Banerji and co-workers investigated how different phases in the BHJ material affect exciton diffusion and charge generation.785,786 A major finding of their work was that it takes about 10–20 ps for carriers to reach the ∼5 nm Electron–hole separation necessary to overcome their Coulomb interaction, in agreement with what previously was found in Ref. 782. Both geminate recombination and polaron pair dissociation was found to be strongly dependent on blend morphology, where creation of percolation pathways for polarons to move away from the interfacial regions increases the yield of free polarons.787 

By combining the results of work on ultrafast charge diffusion and drift782,785,786,788 with work pointing to the importance of delocalized hot charge states for efficient charge separation,776,778 the following picture of charge separation emerges: charge delocalization reduces the electron–hole electrostatic interaction to a level where fast diffusion can separate charges to distances where recombination is inefficient, in only a few picoseconds, and free carriers that contribute to PC are formed in high yield.

Much of the work on charge generation and separation discussed above suggests that relaxed CT states have binding energies significantly larger than room temperature kT, making thermal dissociation at room temperature inefficient. However, there are many possible polymer:fullerene combinations and morphologies, so one should expect CT states to have widely differing properties, e.g., a range of binding energies. The current understanding of CT states was recently reviewed and it was discussed how factors such as the geometry of the D–A interface, electronic polarization, and the extent of electron delocalization affect their nature and influence the radiative and non-radiative decay processes.755 

Exciton dissociation, electron–hole pair (or CT state), and free carrier formation, being the first decisive steps on the route from photon absorption to PC in BHJ organic solar cells, have attracted great interest and excitement of many scientists for almost three decades. We have tried to illustrate the development of this field from the first time-resolved measurements suggesting ultrafast charge generation to increasingly more detailed studies including different materials, and use of different experimental techniques addressing various aspects of the charge carrier dynamics. Historically, describing the processes and involved species a sometimes confusing terminology has been used. For instance, the initially formed photoproduct of polymer exciton dissociation is by some authors called a CT state, some call it a Coulombically bound electron–hole pair and some a CT exciton, or perhaps an exciplex, or why not a polaron pair. All these species can in addition probably exist as hot or thermally relaxed species. In their original definition some of these are certainly distinct entities, but as they are used to describe species in polymer:fullerene photophysics, in many cases, if not all, these species are just different names of the same “entity.” One likely reason for this confusion is that experiments by different groups are performed on different types of samples with often different techniques that probe different physical properties. Studied systems may be complete solar cells, polymer:fullerene blends used in solar cells, model systems for the heterojunction and bilayer structures. It is not surprising if differences in chemical properties and morphology lead to significant variations in the nature and temporal evolution of excited species for such a variety of systems. Nevertheless, we believe that the above discussion illustrates an emerging consensus on the factors and processes that contribute to efficient charge generation and separation in polymer:fullerene BHJ solar cells:

  • A BHJ morphology with large donor:acceptor interface area and sufficiently large (and perhaps ordered) domains that allow efficient delocalization and fast diffusion of charges; at the same time it is necessary to avoid large polymer domains that necessitates long range energy transfer to reach the interface.

  • Avoid low energy localized CT states.

  • Avoid large LUMO energy mismatch between donor and acceptor, i.e., large ET driving energy not required.

This will result in the following charge carrier dynamics:

  • Femtosecond timescale charge generation.

  • Fast and efficient formation of free mobile charges on a ps timescale, effectively intercepting geminate charge generation.

  • Few charges trapped in low-energy CT states that reduce photocurrent yield.

  • Minimized loss of open circuit voltage, VOC.

2. Charge carrier recombination in polymer: Fullerene organic solar cells

In earlier chapters, we discussed the impact of charge recombination for the efficiency of dye sensitized solar cells. As may be expected, recombination is also of key importance for the function and efficiency of organic solar cells. Recombination is the loss process in direct competition with charge extraction and must therefore be minimized by slowing it down. The photogenerated charge pairs (sometimes referred to as CT states) have two options to recombine, either through a geminate process where the electron and hole generated by the same photon absorption recombines, or, after separating into free charges, recombines through a non-geminate process with the charges generated by another photon. This is illustrated in the scheme of Fig. 34.783 

The two processes can be distinguished by their different carrier density (excitation light intensity) dependence. Geminate recombination is independent of carrier density, whereas non-geminate recombination exhibits such a dependence. This is illustrated by the transient absorption kinetics in Fig. 35 monitoring the excitation dynamics from exciton generation to charge recombination. At the lowest excitation fluences, the decay on the ns and longer timescale represents geminate recombination. At higher fluences, the transient absorption decay becomes increasingly faster with increasing excitation intensity [traces (a)–(e)], due to non-geminate recombination. The relative importance (and rates) of geminate and non-geminate recombination has been observed to be strongly material dependent—for some polymer/fullerene blends geminate recombination is relatively fast and completely controls the dynamics even at relatively high carrier density,783 and definitely at the low densities corresponding to solar radiation fluxes. For other polymers and blends, geminate recombination can be very slow and non-geminate recombination plays an important role already at quite low carrier densities.45,774,777,789–792 The kinetics in Fig. 35 show that at the particular excitation intensity used in that experiment, both types of recombination are present and at sufficiently low intensity non-geminate recombination has become too slow to be resolved with the precision of the experiment. In principle, it should be possible to find an excitation intensity (i.e., carrier density) where the two processes are characterized by distinct time decays. However, the strongly non-exponential decay kinetics of both processes can make it difficult to resolve them when simultaneously present. From the point of view of solar cell efficiency, making both recombination processes significantly slower than charge extraction will optimize cell efficiency.

The influence of charge recombination on carrier extraction in polymer:fullerene solar cells was examined in three works on MDMO-PPV/PCBM blends. Nanosecond TA experiment identified a fast <20 ns excitation intensity-dependent phase assigned to recombination between mobile polymer polarons and PCBM anions.792 In the notation used here, this corresponds to non-geminate recombination. A much slower, intensity independent 100 ns–10 ms, phase was also identified and assigned to recombination between low-energy stationary polymer polarons and mobile photogenerated PCBM anions. This must also be considered as non-geminate recombination since the stationary polarons can be generated by continuous background illumination and the negative charges by pulsed excitation light. The latter process was concluded to be of greatest importance for the function of an operating solar cell at ambient solar fluence conditions. In a follow-up work793 the slow, μs-ms, recombination was further studied and it was concluded to be controlled by thermally activated detrapping of the polymer polarons. The low energy polaron sites are filled through continuous illumination and recombination occurs with photogenerated PCBM anions. Increasing the fluence of CW illumination increases the trap filling, which speeds up the recombination. Further studies of the same polymer:PCBM blend794 concluded that simple (Langevin-type) models of non-geminate recombination did not explain the measured dynamics, but a model based on multiple trapping of polarons in a distribution of polymer traps could explain the observed kinetics and its dependence on temperature, light intensity, and PCBM concentration. Under solar illumination, recombination is limited by activation of positive polymer polarons out of deep traps and carrier collection competes successfully with recombination. A number of other works have also shown that non-geminate recombination in organic photovoltaic materials generally is several orders of magnitude slower than predicted by purely diffusive Langevin models. Friend and co-workers have reviewed different non-geminate recombination models (including Nelson's multiple trapping model) as well as experimental work on non-geminate recombination in organic solar cells795 and discussed a number of reasons to the discrepancies between observed non-geminate recombination rates and those predicted by Langevin theory.

Comparing charge recombination times with charge extraction times should be a way to estimate the impact of recombination on solar cell efficiency. Measurements of time-resolved extraction of photogenerated charges in solar cells based on several different BHJ polymer:PCBM materials have shown complete extraction in ≤1 μs.788,796,797 It is not unusual that geminate recombination is faster than this (see, e.g., Ref. 783 and Fig. 35), which would lead to a significant reduction of solar cell quantum efficiency. On the other hand, there are also examples where geminate recombination is much slower than charge extraction.45 The non-geminate recombination on the 100 ns–10 ms timescale for MDMO-PPV/PCBM solar cells mentioned above792 would be on the limit since the ∼100 ns part of the recombination would compete with the extraction. Thus, it appears that both geminate and non-geminate recombination must be considered as potential loss processes in a solar cell device and which of the two is most important is strongly material dependent.

3. Charge carrier photoconductivity and mobility in polymer: Fullerene organic solar cells

Above we gave a picture of charge separation and formation of free charges where charge delocalization and mobility are important properties. Charge mobility is also expected to be essential for efficient transport through the active material for extraction at the electrodes. How carrier mobility contributes to efficient charge separation and generation of free mobile charges that can be extracted as photocurrent we will discuss below. As we described in Sec. II B 1 above, mobility of charged species is directly correlated with conductivity. Therefore, measurement of the transient photoconductivity brought about by the excitation gives direct access to the mobility of charge carriers on the ultrafast timescale. TRTS is the appropriate tool in order to realize this.

Several early TRTS studies on P3HT:PCBM thin films resulted in similar THz photoconductivity kinetics—ultrafast instrument-limited rise, followed by a major fast decay within a few ps, and then a much slower decay on the hundreds of ps to ns timescale of lower amplitude.798–801 The kinetics shown in Fig. 36 are representative for these measurements. The ultrafast rise of the photoconductivity signal was invariably assigned to charge generation, but the few-ps decay was given different explanations—exciton–exciton annihilation,798 interfacial charge transfer,798,801 or electron trapping.799 The slow hundreds of ps decay was, e.g., suggested to reflect charges that survived the fast decay and which could be extracted at the electrodes.798 

Nemec and co-workers investigated two other polymers, APFO3 and LBPP-1, mixed with PCBM. For both samples, the transient photoconductivity was very similar to that discussed above for P3HT:PCBM. However, the interpretation of the fast decay was rather different. For LBPP-1:PCBM the ps decay was assigned to cooling of hot holes, rapidly lowering their mobility.802 For APFO3:PCBM, the photoconductivity spectrum was concluded to be dominated by response from separated charges and bound polaron pairs, and the ps decay was suggested to reflect decrease in mobility of the photogenerated separated charges.803 From these early results, it is clear that the information on carrier mobility was limited, and in cases when measured THz decays were interpreted as decay of mobility it was invariably very fast, on the few ps timescale.

The transient photoconductivity kinetic measurements discussed above, and similar early measurements on conjugated polymers and BHJ materials,46,804,805 were generally obtained with a high excitation density, typically on the order of 1015 photons/cm2 per pulse, in order to achieve a reasonable signal-to-noise ratio. This is due to the fact that the mobility of charge carriers in organic solar cell materials is rather low, on the order of 10−2–10−3 cm2/V s, and the transient photoconductivity is a product of mobility and charge concentration. This complicates the interpretation of photoconductivity kinetics since non-linear effects start to influence the early time processes. Transient absorption kinetics, in the visible spectral region, have shown that at low excitation density (<1013 photons/cm2 per pulse) the onset of charge recombination occurs on the several nanosecond or slower timescale, but at high excitation densities (∼1015 photons/cm2 per pulse) the charge decay is accelerated to a few ps due to polaron pair annihilation.772,773 The distance between photogenerated charge pairs at these excitation densities was estimated to be as small as ∼4 nm. This implies that not much motion of the charges is needed before they meet other charges and can recombine. The sub-ps to few ps timescale on which this process occurs means that it is best characterized as non-equilibrated charge-pair recombination, or annihilation. As can be expected, the process is highly intensity dependent.773 In order to examine if this kind of ultrafast recombination could be responsible for the fast decays observed in THz photoconductivity measurements, time-resolved THz measurements were performed for a TQ1:PCBM blend over a wide range of excitation densities.806 The resulting photoconductivity kinetics are shown in Fig. 37. At the highest pump fluence (1.8 × 1015 photons/cm2 per pulse), the measured kinetics were very similar to that presented in Fig. 36—an ultrafast rise, followed by a few ps decay and a small long-lived signal extending up to 100 ps. As the excitation density is lowered the decay becomes gradually slower, such that at the lowest intensity (9.1 × 1012 photons/cm2 per pulse) the photoconductivity kinetics did not exhibit any decay (within the achieved signal to noise ratio) over 100 ps. An even longer measurement window of up to 400 ps showed that the decay of photoconductivity is on the several hundred ps timescale (Fig. 37). A non-decaying carrier density on the hundreds of ps timescale revealed by optical transient absorption measurements on the same TQ1:PCBM sample,806 at the same excitation intensity, led to the conclusion that the photoconductivity decay in the kinetics shown in Fig. 37 represents decay of carrier mobility on the several hundred ps to ns timescale. It was also noted that this slow decay of carrier mobility is not unique for TQ1:PCBM, but also present in another polymer:fullerene blend, APFO3:PCBM.806 This is several orders of magnitude slower than indicated from the early THz measurements, and shows that a high charge carrier mobility is maintained for times relevant for charge separation and extraction.

With a combination of TREFISH781 and PC measurements807 supported by Monte Carlo simulations the time dependence of charge mobility could be monitored over a much wider time window.788 The results summarized in Fig. 38 shows how mobility of photogenerated electrons and holes in two different BHJ materials (TQ1:PC71BM and TQ1:N2200) during a microsecond decays to the thermalized value due to cooling of the charge carriers. It is interesting to see that the decay of mobility during the first nanosecond as obtained by THz (Fig. 37) measurements is considerably slower than that found by TREFISH (Fig. 38). The reason can most likely be found in the fact that the two measurements probe somewhat different aspects of the mobility—THz very local motion due to the high frequency of the THz field, and TREFISH more long-range diffusion and drift.

4. Charge carrier dynamics in polymer: Non-fullerene-acceptors organic solar cells

As mentioned above at the beginning of this section on organic solar cells, the introduction of non-fullerene-acceptors (NFA) around 2011 and intense development during the following years have implied somewhat of a revival of the field.756–762,765 At the time of writing, the best research NFA organic solar cells have a conversion efficiency over 18% (Refs. 763, 764, and 808) and there seems to be no fundamental reason why with further material optimization PCEs over 20% could not be reached809 and challenge perovskite and other thin film solar cells.

To conclude this section on organic solar cells, we will briefly summarize some recent results on photophysics and charge carrier dynamics of polymer:NFA materials and solar cells. The first few years of work on NFAs was naturally focused on material development and basic characterization, and only more recently some time-resolved and ultrafast work have appeared. A very obvious difference between organic solar cells based on fullerene and NFA acceptors is that NFAs, like those illustrated in Fig. 29, having cores of fused aromatic rings generally absorb light in the red to near-IR spectral region (Fig. 39),810,811 whereas fullerenes absorb at wavelengths shorter than ∼650 nm. This implies that the BHJ polymer donor:NFA acceptor material absorbs light throughout the visible and near-infrared region and the fact that the NFA is the lowest energy absorber will, as we will see below, have consequences for the charge generation process.

In relatively early work, charge generation was studied in a PTB7-Th:PDI polymer:NFA blend using ultrafast transient absorption spectroscopy. It was concluded that ultrafast (<1 ps) long range ballistic electron and hole transfer occurs.812 Ultrafast long range electron transfer has previously been concluded for polymer:PCBM BHJs, and it is now interesting to see that it obviously also occurs for holes. It was considered that this mode of charge transfer requires that charges can access extended regions of delocalized π-band states. Free charge generation in the high‐performance blend of the donor polymer PM6 with the NFA Y6 was thoroughly investigated as a function of internal field, temperature, and excitation energy.813 The results showed that PC generation is essentially barrierless with near‐unity efficiency, regardless of excitation energy. Efficient charge separation is maintained over a wide temperature range, down to 100 K, despite the small driving force for charge generation. Experimental results and theoretical modeling suggested that CT state dissociation is assisted by the electrostatic interfacial field, present between the polymer and NFA domains, which for Y6 is large enough to compensate for the Coulomb dissociation barrier.

The role of morphology in charge generation in polymer(PM6):NFA solar cells was investigated using ultrafast TA spectroscopy with excitation at 800 nm.814 The morphology was varied by mixing-in increasing amounts of low-molecular-weight fractions (LMWF) of the PM6 polymer into the blend. ET from the PM6 polymer to the Y6 acceptor in the blend containing 1% LMWF is ultrafast, <1ps, while in the blend containing 52% of the LMWF there is Förster energy transfer to the acceptor, followed by relatively slow ∼100 ps hole transfer from Y6 to PM6. The reason for the faster electron transfer in the 1% blend was concluded to be the more favorable bulk/interfacial morphological features, realized by closer donor:acceptor (D:A) interactions, smaller D and A domains, and increased D:A interfacial area, facilitating ultrafast electron and hole transfer at the D:A interface.

In a systematic study of three different polymer:NFA blends with a gradually varying energy offset between the S1 state of the NFA acceptor and the CT state of the blend, a deconvolution of different loss processes and a connection to BHJ morphology was established.815 Thus, non-geminate recombination was concluded to be the major loss process competing with charge extraction from the device. Charge generation was concluded to mainly proceed through hole transfer from the NFA acceptor, regardless of which component initially absorbs light. This implies that upon exciting the polymer, the dominating process is energy transfer to the NFA (due to significant spectral overlap between donor and acceptor) followed by hole transfer from NFA to polymer. Structural studies showed that increased interfacial area could be correlated with increased charge generation and higher short-circuit current. Increased local ordering and π–π interaction lead to improved charge transport, favoring efficient charge extraction.

Exciton diffusion length is a measure of how far donor excitons can move to reach the D/A interface where charge generation occurs, and therefore constitutes a constraint to morphology that optimizes conversion efficiency of a solar cell. Similarly, charge carrier diffusion length determines how far photogenerated charges can move to be extracted at the electrodes, and is thereby limiting the thickness of the active layer. Both have been measured for several polymer:NFA materials. Exciton diffusion lengths of several NFAs were measured using exciton annihilation and a technique relying on CuSCN/NFA bilayer devices with variable thickness of the NFA layer for exciton quenching.816 Exciton annihilation has historically been extensively used to determine the size of photosynthetic pigment domains,79 or the number of excitonically interacting molecules in dye aggregates, (see, e.g., Ref. 817) which is analogous information to diffusion length. The measurement of exciton diffusion in NFAs816 focused on NFAs yielding solar cell devices with the highest PCEs reported at the date of the work. The longest diffusion length was found to be 45 nm and more than four times that in the fullerene PC71BM. On the basis of crystallographic data and quantum chemical calculations, the origin to the long diffusion lengths was concluded to be large excitonic couplings, small reorganization energies and small energetic disorder—all due to the stiff conjugated core and aligned transition dipole moments of the NFAs.

There appears to be no ultrafast studies yet published on charge carrier mobility in NFAs or polymer:NFA BHJs. However, to provide some information on this issue, we mention the space-charge limited conduction measurements of thermalized charge carrier mobility and diffusion length performed for PM6:Y6 solar cells.818 Electron and hole mobilities were concluded to be 4 × 10−3 and 2 × 10−4 cm2/V s, respectively. These mobilities and the finding that the measured short circuit current was independent of active layer thickness up to 300 nm led to the estimated electron and hole diffusion lengths of 330 and 70 nm, respectively. Charge recombination at open circuit condition of 10 μs in combination with the long diffusion lengths was considered important properties for the high PCE of a solar cell based on the PM6:Y6 BHJ material. It is interesting to compare the electron and hole mobility of PM6:Y6 with that reported in Ref. 807 and shown in Fig. 38 for TQ1:PC71BM; electron mobilities are about the same, but hole mobility is an order of magnitude higher in the PM6:Y6 blend. Less difference between electron and hole mobility in PM6:Y6 would lead to a more balanced charge transport and facilitate charge extraction.

What ultrafast spectroscopy taught us about organic solar cells

In a very brief summary of this section on organic solar cells, we conclude that ultrafast spectroscopy has provided significant insights into all major steps of PC generation in polymer:fullerene BHJ solar cells, from charge photogeneration to extraction of charges. The mechanism of free charge formation and its relation to BHJ morphology as well as correlations between charge recombination, mobility, and charge extraction appears to be particularly important. The introduction of NFA acceptors has implied a leap in conversion efficiency of organic solar cells. We are probably only at the beginning of a very interesting development with further material development guided by experimental and theoretical characterization, where ultrafast spectroscopy has an important role to play, that may lead to highly competitive organic solar cells.

Organic solar cells—some unresolved issues

  • Little ultrafast spectroscopy work exists on NFA organic solar cells. Charge generation, separation, and recombination are no less important to characterize than in other types of solar cell materials. This is particularly important since existing work has indicated that charge generation may include both initial electron and hole transfer and proceed no matter if the donor or acceptor is excited. Ultrafast work on various polymer:acceptor combinations and morphologies would give insight into material properties controlling charge generation.

  • As mentioned above, charge mobility is poorly characterized in polymer:NFA materials—THz and microwave conductivity measurements would change this.

  • Charge generation, separation, and recombination in polymer:fullerene materials were extensively studied and a possible consensus picture was described above. At the same time, it must be remembered that these processes could depend on the particular polymer:fullerene combination. From a scientific point of view, it could be interesting to investigate more materials. However, from the solar cell efficiency point of view, the interest has shifted to NFA solar cells.

Perovskites are solids described by the formula ABX3, where X is an anion and A and B are cations of different sizes (A is larger than B). Of particular interest here are the OMHPs containing a heavy atom like Pb, a halogen atom (I or Br), and an organic molecule. An OMHP possesses properties of traditional inorganic semiconductors, combined with the great advantage of solution processing. It is the specific crystal structure that leads to a low bandgap energy and high absorption coefficient, allowing a sub-micrometer thick layer to absorb all light in the visible region, which is necessary for solar cell applications.

Initially used as a light-harvesting material in a Grätzel-type solar cell in 2009 with only 3.8% overall power conversion efficiency (PCE),819 the OMHP methylammonium lead triiodide (MAPbI3) has seen unprecedented improvement with a current PCE over 25.5% (https://www.nrel.gov/pv/cell-efficiency.html). This has altered the research landscape in emerging photovoltaic technologies, and these materials have also been investigated in many other related fields, and were for instance discovered to perform well in light emitting diodes,820 as laser materials,821 and for water photolysis.822 Intense research by a large number of groups is pursued in material and device development823–828 as well as various methods of material and device characterization are carried out. Here, we will discuss a few key papers on the ultrafast charge carrier dynamics in OMHPs and OMHPs in a solar cell setting, i.e., OMHPs in contact with charge transport layers. For a recent general review, see Ref. 829 and for more details of many aspects of the ultrafast dynamics, we refer to a few recent reviews.625,830–836

1. Charge carrier dynamics in neat organometal halide perovskites

Excitons, the photogenerated species, in commonly used semiconductors have binding energies on the order of 10 meV, or less, i.e., Si 15 meV, InP 5.1, and GaAs only 4.2 meV.837 Such low binding energies imply that the Wannier–Mott excitons in these materials are stable only at low temperature, while at room temperature (kT ≈ 25 meV) the excitons dissociate on an ultrafast timescale into free charge carries. For the most studied perovskite, MAPbI3, the precise value of the binding energy is still debated, but there is a large body of direct and indirect evidence suggesting it being less than kT at room temperature, approximately 10–15 meV,831,836,838 and a value of only 3.1 meV has been reported for FASnI3 (FA = formamidinium).831 Therefore, for MAPbI3 and many other OMHPs, the majority of excitations at room temperature should result in separated charges. At low temperature, where perovskites undergo structural phase transition, the exciton binding energy is higher831,836,838 and signatures of Wannier–Mott excitons have been observed.839,840 The function of a perovskite material in photovoltaic and optoelectronic applications is dependent on the formation, mobility, transport, and recombination of charges. The generation yield and the fate of the mobile charges can lead to success or disaster to a promising solar cell technology.

Various ultrafast methods have been used to report on different aspects of perovskite carrier dynamics. Transient absorption or photoluminescence in the visible part of the spectrum can be used to probe band-to-band transitions and luminescent species. TRTS and TRMC are particularly powerful methods that give direct insight into properties and dynamics of photogenerated mobile charges. For MAPbI3 thin films at room temperature, charges with a combined electron and hole mobility of ∼20 cm2 V−1 s−1 were seen to be formed on the few-ps timescale after light absorption841,842 (Figs. 40 and 41). In Ref. 841, individual mobilities for electrons and holes were also reported and obtained by first measuring the total e + h mobility (∼20 cm2 V−1 s−1) and then the hole mobility (7.5 cm2 V−1 s−1) by removing the electrons from the perovskite film by injection into nanostructured TiO2 (Fig. 40). This shows that electron and hole mobility in MAPbI3 are very similar, which prevents creation of built-in electric fields and space charge-limited PC that lowers PCE. Mobilities (e + h, e, and h) for several different OMHPs thin films and single- and poly-crystalline samples measured by various techniques (THz, microwave conductivity, photoluminescence quenching, Hall effect, etc.) are summarized in the review by Herz.830 Measured values vary from ∼1 to ∼2000 cm2 V−1 s−1, depending on type of perovskite and preparation (film/crystal), but even for the same perovskite and type of preparation there is significant variation between different studies. This shows that factors related to sample preparation (morphology, impurities, etc.) and measurement technique also contribute to the spread in reported mobility values. Carrier mobility of OMHPs was also found to have a negative temperature dependence, i.e., mobility increases with decreasing temperature.830,843

Long lifetime of the photogenerated charges, i.e., slow recombination, is essential to allow sufficient time for extraction of the charges. Time-resolved THz spectroscopy and time-resolved microwave conductivity, both directly report on the population and mobility of charges. However, it has to be remembered that the measured conductivity is a product of mobility and carrier concentration. Thus, in order to resolve the contribution of the two quantities to an observed decay of conductivity, independent measurements of the time dependence of one of them is required. By combining TRTS and optical transient absorption measurement on MAPbI3 films it was shown that carrier mobility remains constant on the ns timescale.841 TRMC extends the accessible timescale into the microsecond range, and it was shown that geminate charge recombination in MAPbI3 at room temperature proceeds on the 5 μs timescale and longer841,843 (Fig. 41). Time-resolved PL measurements also suggested long recombination times,844 albeit somewhat shorter than those obtained from TRMC, and it was speculated what the reasons to the difference could be.843 The non-geminate recombination was seen to be several orders of magnitude slower than predicted by Langevin theory (i.e., diffusion limited) due to a temperature activated recombination of the encounter e–h complex preceding the actual recombination.842–844 The long recombination time and relatively high mobility in MAPbI3 translate into a diffusion length of charges well over 1 μm (Refs. 841 and 843–846)—sufficiently long to efficiently extract charges out of the active material in a thin film solar cell.

The typical total (e + h) mobilities observed for thin film perovskites (∼1–50 cm2 V−1 s−1) and single crystals (∼50–500 cm2 V−1 s−1)830 are several orders of magnitude higher than in for instance organic semiconductors,806,847 but still much lower than the electron mobility in traditional inorganic semiconductors like GaAs (9400 cm2 V−1 s−1).837 The reason to the difference between thin film and single crystal perovskite can probably be found in non-material specific, “extrinsic” factors830 like size of microcrystallites, grain boundaries, defects, and impurities; but, why is the mobility in a single crystal OMHP so much lower than in for instance GaAs? This fact combined with the carrier dynamics characteristics described above, long recombination time,841,843–846 negative temperature dependence of mobility,830 led to the hypothesis that the charge carriers in OMHPs are best characterized as large polarons and their dynamics as a result of interactions between electrons (holes) and the electrical field generated by collective longitudinal optical (LO) phonons.848 Calculations of the so-called Fröhlich coupling849,850 based on LO phonons yields a carrier mobility on the order of 100 cm2 V−1 s−1, similar to measured values in single crystal OMHPs (see, e.g., Refs. 851 and 852). A visualization of the electron–phonon coupling is a quasi-particle polaron, which can be pictured as a charge surrounded by a distorted crystal lattice. As the charge diffuses through the crystal, it drags along with it the deformation of the crystal lattice, slowing down the mobility from that of the bare charge. The magnitude of the Fröhlich coupling constant determines the size of the polaron and calculations suggest a radius of ∼5 nm in OMHPs, i.e., several times the crystal lattice constant, consistent with a Fröhlich large polaron (see, e.g., Refs. 830 and 853 and references therein).

The large-polaron hypothesis in OMHPs848 triggered a lively experimental and theoretical activity in order to substantiate the hypothesis (see Ref. 831 for a review of some of this work). The negative temperature dependence of mobility mentioned above was by several authors seen as an indication of electron-LO-phonon scattering843,854–857 (Fig. 42). Several of these studies observed an approximate T−1.5 dependence of the carrier mobility, while calculations851 predicted a T−0.46 dependence. Authors have also suggested a scattering mechanism involving electrons and acoustic phonons,858 but as we will see below, despite some shortcomings there seems to be a growing consensus that electron-LO-phonon interaction best describes charge carriers in OHMPs. In order to resolve the limitations of the Fröhlich interaction model, there is an ongoing discussion if additional effects need to be considered for a complete description of the charge carrier behavior. It is beyond the scope of this review to give a detailed description of these considerations, but the review of Herz831 provides a concise discussion. In short, anharmonicity of vibrations, not taken into account by the Fröhlich model may modify the results of the calculations; studies of couplings between organic cations and the lead-halide sub-lattice as well as the temperature-dependence of the dielectric response across a wide frequency range, may help to elucidate these issues. Finally, it should be mentioned that the temperature dependence of the carrier mobility, signaling disagreement between experiment and the Fröhlich model, is not the same for all OMHPs and may vary if measured over a wide temperature interval.831 Thus, for MAPbI3 several studies report a T−1.5 dependence, while for FAPbI3 a close to T−0.5 dependence has been reported.831 It has been suggested that the reason for this difference between perovskite materials could be due to extrinsic effects, i.e., differences in morphology and crystallinity that could represent barriers to charge carrier diffusion.831 Measurements of carrier mobility temperature dependence of phase pure materials of several different perovskites could help to resolve these issues.

Additional evidence for the polaron picture was obtained from measurements of the rise of THz conductivity of three different OMHPs (MAPbI3, FAPbI3, and CsPbI3) after photoexcitation with a 35 fs pulse of varying photon energy (700–400 nm)853 (Fig. 43). An ∼1 ps rise of conductivity was observed, similar to that observed in several other works (see, e.g., Refs. 806 and 840) and based on the temperature and excitation excess energy dependence of the conductivity rise it was interpreted to be a result of a consecutive process of polaron formation and carrier cooling. The polaron formation occurred with a temperature independent time of ∼400 fs, almost the same for all three OMHPs. Carrier cooling was seen to be both temperature and excess energy dependent with a maximum time of ∼1 ps (Fig. 43).

Signatures of large polaron formation in OMHPs discussed above are their kinetic characteristics as well as their temperature dependencies. A next step in substantiation of the large polaron picture would be to obtain spectral fingerprints of the LO phonon–charge interaction. This was achieved in several works. Optical pump-THz probe ultrafast measurements of photoconductivity were seen to be superimposed with phonon frequencies, which were affected by the photogenerated charges.859 The phonon absorption bands, initially blue-shifted on a sub-ps timescale following charge generation, red-shifted on a slower timescale when charge density decays (Fig. 44). This was taken as fingerprint evidence for electron–phonon coupling and polaron formation in CsPbBr3 nanocrystals. The time for polaron formation was similar, ∼400 fs, to that observed in Ref. 853 (from measurement of the rise of THz conductivity), and with the help of DFT calculations Pb-Br-Pb bending modes were shown to be involved in the polaron dynamics. Ultrafast optical Kerr effect and reflectance measurements on single crystal MAPbBr3 and CsPbBr3 provided further evidence for polaron formation.860 The optical Kerr effect measurements probe the response of the crystal lattice to carrier generation, while the reflectance measurements probe the dynamics of the electronic degrees of freedom. Both responses resolved a fast time constant, 0.3 ps for MAPbBr3 and 0.7 ps for CsPbBr3, and was interpreted as formation of a large polaron following photogeneration of charges. First principle calculations supported the experimental results and identified the Pb–Br–Pb deformation modes as responsible for the polaron formation, as well as explained the difference in polaron formation time of the two perovskites. Nuclear wave packet dynamics of low frequency modes at 20, 43, and 75 cm−1, assigned to bending and c-axis stretching modes of the Pb-I-Pb bonds, were also seen as signature of polaron formation in MAPbI3 thin films.861 On the theoretical side, assuming that the charge carrier is a large polaron, an upper limit of the mobility was estimated using Feynman polaron theory and measured values of Br-Pb-Br bending and stretching modes of MAPbBr3.852 Mobilities of 83 cm2 V−1 s−1 for electrons and 265 cm2 V−1 s−1 for holes were obtained, in reasonable agreement with measured values for single crystals. From this discussion it appears to be an increasing consensus that the large-polaron picture, through the Fröhlich mechanism,849,850 best describes charge carrier dynamics in OMHPs.

a. Hot carrier dynamics

Hot carrier solar cells have the potential to increase power conversion efficiency above the Shockley–Queisser limit, but no useful material has so far been found due to fast and efficient electron–phonon scattering that dissipates the excess electronic energy. For MAPbBr3 single crystal hot fluorescence with lifetime of ∼100 ps was observed, signaling long lived hot electrons.862 It was suggested that dynamic screening by a solvation process or large polaron formation protects the energetic carriers from cooling. Similar observations were done for MAPbI3 thin films, more relevant for solar cell applications.863 In this case, fast initial cooling with a time constant of 0.25 ps was observed, followed by much slower cooling leaving ∼0.25 eV of excess energy unrelaxed for ∼100 ps. At high carrier densities cooling may be slowed down through hot-phonon bottlenecks; for MAPbI3 films cooling slowed down from ∼30 fs to ∼30 ps when increasing carrier density by a factor of ten above 5 × 1017 cm−3.864 This bottleneck was suggested to be associated with distortions of the Pb-I-Pb lattice as detected by a long lived (∼10 ps) 0.9 THz phonon oscillation in 2D electronic spectroscopy of MAPbI3 films.865 In another 2D electronic spectroscopy study, thermalization of photogenerated charges in the MAPbI3 perovskite was studied. It was found that cooling of photogenerated carriers occurs in two steps—first formation of a hot thermalized population on the <100 fs timescale followed by cooling on the several hundred fs timescale.866 Thermalization was concluded to occur through electron–electron scattering and cooling through electron–phonon scattering. It was argued that hot carrier extraction in a perovskite solar cell under continuous illumination will be difficult because the hot photogenerated carriers will very rapidly, <100 fs, be thermalized with a large background population of cold (300 K) carriers, without a significant change of temperature of the total population. In this way, the excess energy of photogenerated charges will be rapidly lost (Fig. 45).

2. Charge carrier dynamics of perovskite/transport layer devices

In a solar cell, transport layers are used to transport electrons and holes from the light absorbing (perovskite) layer to electrodes. With an origin as a sensitizer in DSCs,819,867 it was natural to interface the perovskite sensitizer to TiO2 for electron capture and transport.823,826,846 Later, organic materials have been used both for electron and hole transport. Two of the most preferred organic choices are PCBM as electron transport material,868–870 while spiro-OMeTAD {2,2′,7,7′-Tetrakis[N,N-di(4-methoxyphenyl)amino]-9,9′-spirobifluorene} is used as HTM.823,826–828,867 In addition, these layers may slow down charge recombination and decrease the concentration of trap states. For a well-functioning device, charges need to be efficiently transferred from the light absorbing perovskite to the charge transport layers. We will give a few examples of work to characterize these processes and start with hole injection into HTMs.

In Ref. 871, hole injection in a MAPbI3/spiro-OMeTAD bilayer device was studied using time-resolved THz spectroscopy. Spiro-OMeTAD has been demonstrated as a good hole transporting material, i.e., most of the best performing perovskite solar cells have used it or some derivative as HTM,872 despite its very low conductivity, 10−8 S/cm.873 However, its valence band has a 0.57 eV difference with respect to the valence band of perovskite,874 highly favorable for charge transfer. In the MAPbI3/spiro-OMeTAD bilayer device, an instantaneous rise of the THz signal to a mobility three times less that observed in neat MAPbI3 was interpreted as ultrafast (≪1 ps) hole injection from perovskite to spiro-OMeTAD (Fig. 46); the holes with their low mobility do not contribute to the measured photoconductivity and the observed mobility is due to electrons remaining in the perovskite layer. Fast (<1 ps) and efficient hole transfer from perovskite to spiro-OMeTAD was also concluded from several works using more indirect optical transient absorption measurements.867,875–877 Optical TA spectroscopy was also used to monitor hole injection into another organic polymer, PEDOT:PSS, and similar to spiro-OMeTAD it was found to occur on the sub-ps timescale (<200 fs).878 Another possibility for a HTM is to explore the use of inorganic hole conducting semiconductors with high mobility, high transparency in the visible region, and good chemical stability. Inorganic HTMs, e.g., NiO, CuI, and CuSCN, were used and have demonstrated high PCEs.879,880 This demonstrates the potential of using, e.g., NiO as an effective inorganic hole extractor. THz photoconductivity kinetics were obtained for MAPbI3/NiO to conclude that holes are injected into NiO on the ultrafast sub-ps timescale.881 

Next, let us turn to injection of electrons from perovskite to an electron transport material. Electron injection from perovskite to TiO2 was found to occur on the sub-ps timescale, both from time-resolved THz871 (see Figs. 40 and 41) and optical TA measurements,877 similar to what was found for many DSC materials.625 Electron injection from perovskite to PCBM appears to be more complex, and there is some variation in reported injection times. For a MAPbI3/PCBM double layer, the transient THz photoconductivity exhibited an instantaneous rise, to the same amplitude as in neat MAPbI3, followed by a partial ∼100 ps decay (Fig. 46). As a result of the low electron mobility in PCBM (0.005 cm2 V−1 s−1)847 and high hole mobility in the perovskite (10 cm2 V−1 s−1), the ∼100 ps decay was interpreted as a combined result of (relatively slow) electron injection from perovskite to PCBM, and recombination between highly mobile holes in the perovskite and injected electrons pinned at the MAPbI3/PCBM interface.871 Several studies of electron injection from perovskite to PCBM using femtosecond optical TA have been reported. In Ref. 878, electron transfer was concluded to proceed over an extended timescale from ∼1 ps to many picoseconds and even nanoseconds, thus similar to what was observed in the THz study by Ponseca et al.871 Another TA study found the injection to be even slower, characterized by a rate constant of 0.61 ns−1.882 Still another ultrafast TA study combined with transient reflectivity measurements showed that perovskite to PCBM CT occurs on the sub-ps to ∼10 ps timescale depending on the perovskite film thickness. One possible explanation to this variation in reported injection times is that it in fact proceeds over a wide time window, and perhaps depends on methods of sample preparation. Another possibility is what was suggested in Ref. 871, that the measured kinetics is a combined result of electron injection and charge recombination at the perovskite/PCBM interface.

What ultrafast spectroscopy taught us about organometal halide perovskite solar cells

Ultrafast spectroscopy has highlighted a number of key features and properties of OMHP materials and devices based on OMHPs as active material.

  • Charge photogeneration in most neat perovskites is ultrafast and occurs on the sub-ps timescale at room temperature, due to low exciton binding energy (∼10 meV) at room temperature.836,838

  • Charge carrier mobility in OMHP thin films on the order of 10–20 cm2 V−1 s−1 (and ∼100 cm2 V−1 s−1 for single crystals) is several orders of magnitude higher than in organic semiconductors, but still much lower than in traditional inorganic semiconductors like GaAs.

  • The relatively high mobility translates into slow charge recombination (>5 μs) and long diffusion length (>1 μm).

  • Charge carrier mobility has a negative temperature dependence, i.e., becomes higher at lower temperature.

  • These characteristics of OMHP excitations can be rationalized with an electron-LO phonon model and large polaron picture.

  • In devices where perovskite layers are contacted with charge transport layers, electron and hole transfer often occurs on the ultrafast, <1 ps, timescale, and in any case much faster than charge recombination.

Organometal halide perovskite solar cells—some unresolved issues

  • The charge carrier characteristics of OMHP materials have been rationalized with an electron-LO phonon model and large polaron picture, although some features, e.g., the temperature dependence of carrier mobility, do not agree with that predicted from theory for all studied OMHPs. There is an ongoing discussion of possible additional effects that could contribute to close the gap between experiment and theory (see discussion above and Ref. 831).

  • Non-Pb perovskites give solar cells with potentially lower toxicity, but with much lower efficiency than cells based on Pb-perovskites, and much less ultrafast work has been performed on the Pb-less materials. More work on the carrier dynamics of Pb-less perovskites may lead to better understanding of the reason for this difference, which could help producing more efficient solar cells.

  • Like for most other solar cell technologies, most ultrafast work is performed on neat OMHP materials, and much less on whole cells or OMHPs in contact with charge transport materials. Thus, studies of the carrier dynamics associated with the OMHP/charge transport material interfaces could provide additional understanding of the solar cell function.

1. Background and basic principles

The extraordinary success of natural photosynthesis discussed in the first part of this review has provided an inspiration for modern molecular sciences to design other molecular systems and materials capable of harnessing solar energy to generate solar fuels.16,883,884 Instead of relying on evolution of biological systems, molecular and materials approaches for solar fuels production are based on advances toward chemical conversion processes in the field of photocatalysis.885–887 A particular challenge for most solar fuels approaches is the difficulty to generate energy-rich fuel products such as gaseous hydrogen (H2) or liquid ethanol (C2H5OH) from low-grade and inert small molecule feedstock materials such as water (H2O) and carbon dioxide (CO2). Thus, a significant part of the solar fuels challenge is that the harvested light energy must be utilized in an efficient manner to store energy in the chemical bonds of the solar fuel products.

Natural photosynthesis can in principle be utilized indirectly through the energy content stored in the generated biomass according to the famous textbook formula for photosynthesis, emphasizing the generation of carbohydrates (CH2O) from carbon dioxide and water:

nCO2+nH2O+photons[CH2O]n+nO2.

Molecular approaches for artificial photosynthesis, in contrast, often focus on the direct light-driven generation of chemical energy from the splitting of one water molecule into its oxygen (1/2 O2) and hydrogen (H2) constituents. A classical photocatalytic scheme that accomplishes a comprehensive solar fuels approach is the combination of a light-harvesting PS unit with a water splitting catalyst on one side and a proton reduction catalyst on the other side, as shown schematically in Fig. 47. The photocatalytic conversion proceeds through successive photoinduced ET steps as illustrated in Fig. 47, where an important challenge to meet is to achieve multi-ET in order to accomplish the complete bond breaking and bond making processes involved in the chemical conversion. In the case of the water splitting illustrated in Fig. 47 this requires the transfer of four electrons per water molecule according to the half-reactions given by the two equations below:

2H2OO2+4H++4e,
4H++4e2H2.

The solar fuels field has grown rapidly over the last few decades and now covers a vast range of homogeneous and heterogeneous photochemical approaches to generate a wide array of high-energy fuels.888,889 Ultrafast studies of key catalytic processes in the field of solar fuels are largely of newer date and not so numerous.886,890–892 Here, we highlight knowledge gained by ultrafast spectroscopy with the help of some recent examples.

Ultrafast solar fuels research concerns at least three different general aspects that together form a framework for understanding the type of system under consideration: the targeted type of solar fuels conversion, the catalytic material used, and the type of photoinduced processes involved.625 At the heart of the ultrafast aspects of any photocatalytic conversion processes are light-harvesting and photoinduced CT processes, which in many cases where the full chemical conversion is not formed on ultrafast timescales will be followed on slower timescales by charge transport and molecular diffusion processes. Slower chemical conversion steps of photochemically activated species that can include electrochemical oxidation and reduction processes as well as slower bond making and bond breaking processes are generally also present, but not considered in this review.

The main solar fuels reactions include (1) hydrogen evolution reactions (HER), (2) water splitting catalysis including oxygen evolution reactions, as well as (3) CO2 reduction to a variety of high-energy products such as methanol (CH3OH) and formic acid (HCOOH). From a broader perspective, there are also other high-grade targets including conversion of nitrogen gas to ammonia as well as a range of other high-energy small molecules and organic fuels. For each of these conversion processes, a variety of molecular, supramolecular, and heterogeneous systems have been or are developed. It is easy to see that these three fundamental reactions are by necessity closely intertwined in a way that they clearly cannot be presented without a lot of overlap. In this section, we present a progression of key ultrafast spectroscopy studies of fundamental photoinduced processes relevant for many photocatalytic conversion processes, using some of the most promising catalytic systems. We start from relatively simple model systems and proceed toward functionally optimized complex materials, accepting that a full account of everything that has been done in this field falls well beyond the scope of the present exposition.

2. Molecular photocatalysis

Photocatalysis is typically initiated by excitation of a PS unit to generate a high-energy excited state capable of driving subsequent catalytic steps. This is illustrated schematically in Fig. 48 for the common case of a photoredox cycle that involves oxidative and reductive ET reactions between the PS and electron donors (D) and acceptors (A). Here, the completion of the PS photocycle requires both an oxidative and a reductive step, but the order between these steps can differ as shown in Fig. 48. The photoinduced reaction mechanisms and rates depend a lot on the system composition. In the simplest case, the photosensitizers are selected based on their excited state redox potentials to drive a specific photoreduction or photo-oxidation reaction with sacrificial electron donors and acceptors used to complete the cycle. In more sophisticated systems separate light-harvesting and catalytic units can be coupled. In such cases, the excited photosensitizers are used to reductively or oxidatively activate a designated catalyst that would then take the role of the donor or acceptor in the initial photocycle in Fig. 48 that couples to a redox-catalysis cycle.

The photocycle in Fig. 48 provides insight to how time-resolved spectroscopy can be used to elucidate important mechanistic aspects of photocatalytic processes.893 The excited state of many of the most widely used photosensitizers, such as Ru(II), Ir(III), and Cu(I) complexes, can be monitored by time-resolved photoluminescence (TR-PL) measurements. By monitoring the PL decay, for example, by TCSPC technique (see Sec. II B 2), the bimolecular quenching of the PS excited state can be tracked as part of a classical Stern–Vollmer analysis of bimolecular quenching characteristics. Moreover, this allows the distinction between oxidative and reductive quenching mechanisms, i.e., determining whether the initial ET step is due to interaction of the PS with an electron acceptor or donor. Such quenching studies are, however, limited to monitoring the initial luminescent state of the PS, but further identification of reactive intermediates and long-lived products can be accomplished using time-resolved optical absorption spectroscopy. This includes transient absorption for investigations of ultrafast processes on fs–ps timescales, as well as flash photolysis for the formation and decay of ns and longer-lived photoproducts. These time-resolved optical absorption spectroscopy techniques are particularly useful when there are clear spectroscopic bands that allow facile identification of different states of both the photosensitizer and other reacting species. These standard time-resolved optical spectroscopy techniques for photochemical investigations are increasingly also complemented by time-resolved spectroscopy using a wider range of probe frequencies to provide complementary information. This includes, e.g., a growing range of time-resolved x-ray spectroscopy techniques capable of probing both structural dynamics and local electronic structure (such as the spin/oxidation state of a metal center),41 as well as time-resolved IR spectroscopy to probe changes in molecular bonding characteristics,894 and time-resolved terahertz to probe photoinduced conductivity with high time-resolution.714 In many cases theoretical support can also be obtained from quantum chemical calculations and excited state dynamics simulations. Together, the combination of several experimental and theoretical techniques provide good opportunities for increasingly comprehensive characterizations of photophysics and photochemistry in complex photocatalytic systems. The role of optical spectroscopy to characterize key light-driven steps have been reviewed in significant detail,625 including recent reviews by Kandoth et al. and Päpke et al. that place the optical spectroscopy in the full context of photoredox catalysts.893,895

A wide range of classical organic photochemical transformations can be accomplished in solution,617 and ultrafast dynamics of photocatalytic reactions including light-harvesting dynamics and photoinitiated bimolecular processes have been extensively reviewed.896 The organic photochemistry field has provided a lot of general and fundamental understanding of light-driven photochemical conversion processes in solution. This includes classical issues of driving force and diffusional dependence of photochemical quenching, as well as fundamental insight into more elaborate aspects of photoinduced bimolecular reactions such as Sumi–Marcus behavior for ultrafast reaction rates in high electron donor concentrations.896,897 However, the purely organic systems largely falls outside the main focus for many of the most actively investigated solar fuels applications based on coordination complexes, as well as heterogeneous and molecular-photoelectrode hybrid systems discussed more extensively in Secs. IV F 3–IV F 6.

3. Transition metal-based photocatalytic systems

Photoactive transition metal complexes have played a particularly prominent role for solar fuels related photocatalytic applications due to a favorable combination of long-lived excited states and superior ability of the metal center to participate in photoinduced oxidation and reduction (photoredox) processes with maintained chemical integrity.898 Moreover, their versatile redox and metal-ligand bonding properties make transition metal complexes generally useful for a wide range of catalytic application that also includes conversion steps that are not light-driven, altogether making them a natural cornerstone of many photocatalytic processes. The role of optical spectroscopy to investigate various mechanistic aspects of the operation of photoredox catalysts was recently reviewed.895 

Examining ultrafast excited state relaxation of transition metal complexes following initial photoexcitation is a natural starting point for understanding the ultrafast dynamics of many photocatalytic systems where time-resolved optical spectroscopy often plays a key role for the mechanistic characterization of excited state dynamics.625 Further additions of increasing value for comprehensive excited state dynamics characterization comes from time-resolved x-ray spectroscopy41,899 as well as advanced computational studies.900,901 Below, ultrafast aspects of key types of excited state photochemistry/photophysics are discussed. The types of photophysics discussed here involve MLCT, LMCT, MC, and ligand-centered photoactive states.

a. Metal-to-ligand charge-transfer photocatalysts

Early efforts to develop molecular photocatalysts relied heavily on light-harvesting coordination complexes with long-lived excited states of Ru(bpy)3 and related octahedral d6 polypyridyl complexes of the second and third row transition metals. As introduced above, many such complexes exhibit nanosecond or longer excited-state lifetimes suitable to overcome diffusion timescale limitations for bimolecular reactions in solution.902 These complexes rely extensively on the favorable photophysical properties of MLCT excited states that are illustrated in Fig. 49 for the classic case of excitations from the occupied t2g levels on the metal to unoccupied π* orbitals on the ligand in octahedral d6 complexes.

A wide array of photoactive second- and third-row transition metal ions of, e.g., Pt and Ir with similar photophysics, principally involving long-lived 3MLCT states commonly employed in molecular photocatalytic applications, have been investigated for photocatalytic applications, including also that they to varying degrees have been subject to corresponding photophysical and photochemical studies down to ultrafast timescales.620 From a photocatalytic perspective the interest in many photoactive light-harvesters with long-lived excited states, however, naturally focuses on subsequent dynamics for driving electron or excitation energy transfer processes (see below) rather than on the largely stable nature of the CT excited state that develops within the first few ps and then persists for the ns/μs excited state lifetime. In contrast, it can be noted that the long-lived excited state lifetime and in particular, luminescence properties of many light-harvesting complexes of Ru, Ir, Pt, etc., are of vital interest to other related applications such as light-emitting devices, biosensors, and photodynamic therapy.903 Promisingly, a variety of coordination complexes, such as Cu(I) complexes, with a d10 electronic configuration are emerging as viable alternatives for a range of MLCT-based photochemical applications using an abundant first row transition element.659,752

b. Ligand-to-metal charge-transfer photocatalysts

Many coordination complexes with easily oxidized ligands and readily reduced metals exhibit ligand-to-metal CT excitations that constitute a fundamental reversal of the charge transfer compared to the MLCT complexes discussed above. The electronic structure of such LMCT excited states featuring a hole on the ligand typically provides for strongly photo-oxidizing coordination complexes suitable for driving a wide range of energetically challenging oxidation reactions such as water oxidation. It can be noted that such complexes capable of oxidizing regular and halogenated hydrocarbons can also be utilized for waste disposal and other photochemical applications.620 However, with much fewer complexes known to exhibit favorable excited state photophysics with long-lived and emissive LMCT excited states much less is also known about the ultrafast dynamics in these complexes compared to more widely explored MLCT-based photosensitizers of Ru(II), etc., discussed above.904 

Kirchhoff and co-workers demonstrated that Re(II)(dmpe)3 [dmpe = 1,2-Bis(dimethylphosphino)ethane] complexes with a doublet 5d5 ground state electronic configuration displayed a rare case of luminescence from doublet 2LMCT excited states with nanosecond lifetimes.647 Here photophysics is fundamentally different from the more widely used d6/3MLCT light-harvesting complexes discussed above in at least two ways. First, because of the orbital occupation with just one unpaired electron in both ground and excited state, the excited CT state cannot undergo intersystem crossing to a lower energy CT excited state and thus the direct decay to the ground state is spin allowed for both radiative and non-radiative deactivations. Second, the excitation involves the creation of a hole in a normally occupied ligand orbital rather than the population of a normally unoccupied ligand orbital by an electron. Understanding the implications of these unusual properties more broadly will be important for further photocatalytic exploitations. The strong photo-oxidizing power of such 2LMCT excited states was exploited by Sullivan and co-workers to demonstrate photoinduced oxidation of a series of organic donor species with bimolecular quenching studies providing insight into the driving force dependence of the bimolecular ET accompanying the excited state quenching.905 

The recent report of a discovery of a Fe(III)-NHC complex with bis-imidazol ligands displaying favorable excited state lifetimes (∼>100 ps) and photoluminescence from a low-spin 3d5 electronic configuration by Chábera et al. in 2017 paved the way for the development of a wider spectrum of photoredox chemistry using earth-abundant Fe(III) coordination complexes.645 These Fe(III) complexes constitute an isoelectronic analog to the Re(II) complexes discussed above. In 2019, Kjær et al. presented a new Fe(III) hexa-NHC complex with significantly improved excited state lifetime (∼2 ns) and PL quantum yield of ∼2% for the 2LMCT state.650 The ns lifetime together with favorable excited state oxidation and reduction potentials allowed for the demonstration of both photo-oxidation of diphenylamine (DPA) as a prototype organic donor, and photo-reduction of methyl viologen (MV2+) as a common acceptor, using flash photolysis photoproduct detection on nanosecond timescales.

While the significant excited state photoredox potentials make this complex interesting as an earth-abundant photosensitizer for photocatalytic conversions,751 the 2 ns excited state lifetimes is still comparatively short and potentially is a limitation for slow diffusion controlled bimolecular photoredox reactions in solution. This diffusion limitation was overcome in a recent investigation of the full photocycle of the photosensitizer in electron donating solvent environments by studying the dynamics of charge separation and recombination down to ultrafast timescales. Fe(III) complexes immersed in solvent mixtures up to high electron donor concentrations (>1 M) for the liquid organic donors triethylamine (Et3N) and dimethylaniline (DMA) were studied using femtosecond transient absorption spectroscopy, as a means to investigate intrinsic electron dynamics in close contact configurations that avoid the usual diffusion limitations (Fig. 50).906 Similar to what has previously been investigated in organic model donor–acceptor systems, ultrafast bimolecular ET dynamics on the ≲1 ps timescale was observed for both oxidative quenching of the photosensitizer excited state and subsequent reductive ground state regeneration (Fig. 50). These ultrafast bimolecular ET rates outpace typical solvent reorganization dynamics in the classical Marcus ET model, indicating that the bimolecular ET proceeds by a more elaborate activation process involving additional reaction coordinates such as intramolecular reorganizations as introduced in the Sumi–Marcus model.906 

While the efficient initial charge separation is promising for driving photochemical reactions with these kinds of 2LMCT complexes, the original results presented in the proof-of-principle full photocycle study in acetonitrile solvent also showed ultrafast charge recombination, effectively outpacing solvent cage escape of the charge separated photosensitizer and electron donor. This severely limited yields of photoproduct formation.906 This issue was recently addressed by Ludowic and co-workers who studied photoredox chemistry of the same complex in different solvents. In particular, significantly improved cage escape and resulting high yields of photoproduct formation was demonstrated in a low-polarity dichloromethane solvent (Fig. 51). Visible-light-mediated dehalogenation in CH2Cl2 using the [Fe(III)(phtmeimb)2]+ photosensitizer in the presence of triethylamine (TEA) as sacrificial electron donor (A) was studied by nanosecond and femtosecond transient absorption as well as by time-resolved infrared spectroscopy. Reductive ET between the Fe(III)-photosensitizer and DMA was experimentally confirmed, with ket = 2.3 × 1010 M−1 s−1 generating a reduced Fe(II) species that could further reduce the substrate 2a, with k = 1.6 × 108 M−1 s−1 (Fig. 51).907 

Another aspect of Fe(III) complexes relevant for photocatalytic applications is that they can undergo pairwise photoinduced charge disproportionation under favorable interaction conditions that includes bimetallic complexes, or other close contact arrangements, such as high photosensitizer concentrations. This is well-known, for example, in dimeric heme-nonheme Fe(III)–Fe(III) complexes,908 where excitation of one of the Fe(III) centers results in significant electron transfer on a faster timescale than the competing intramolecular deactivation dynamics according to the basic reaction scheme:

Fe(III)+Fe(III)+hvFe(III)*+Fe(III)Fe(II)+Fe(IV).

Such charge disproportionation reactions are possible for metal centers, such as Fe(III), that are simultaneously both easily oxidized and easily reduced. The reaction is interesting since it provides a self-interaction potentially capable of generating both long-lived oxidized and reduced metal centers of interest for redox-catalysis. For example, heme-nonheme Fe(III) dimers have been shown to drive photo-oxidation and atom transfer reactions following light-induced charge disproportionation.908 Recently, Kaul et al. investigated charge disproportionation in concentrated solutions of [Fe(phtmeimb)2]+, where phtmeimb is {phenyl[tris(3-methylimidazol-1-ylidene)]borate} using time-resolved optical spectroscopy to identify the Fe(IV) and Fe(II) products.909 

c. Metal centered photocatalysts

Several types of transition metal complexes feature photoactive metal centered ligand field states that are interesting for photochemical applications.910 The Laporte forbidden nature of d–d transitions constitutes a double-edged sword for photochemical applications. On one hand, the weak coupling between the ground and excited states can play a beneficial role to achieve long-lived excited states even down to the near-IR region. On the other hand, direct d–d photo-excitation is often correspondingly weak, so the d–d transitions do not lend themselves well for efficient light-harvesting. Therefore, practical utilization of d–d excited states for photochemical applications is often achieved through higher energy excitation of stronger ligand-centered or CT excitations, followed by relaxation to a lower excited d–d excited state in line with Kasha's rule that the lowest excited state is typically responsible for the photo-functionality of the complex.

Several Rh(III) polypyridyl-type complexes such as [Rh(III)(phen)3]3+ (phen = phenanthroline) are known to display long-lived photoluminescence from metal centered ligand field states, which for a 4d6 transition metal complex typically originates from an 3[(t2g)5(eg)1] electronic configuration that can be populated by intramolecular excitation energy transfer from an initial excitation of a ligand-centered π-π* state.911 As the ligand field splitting is much smaller in the first row (3d) transition metal complexes owing to the primogenic effect,912 achieving corresponding photoactive states of sufficient excited state energy in an analogous first-row Co(III) complex has been very challenging. Recently, the utilization of the strong sigma-donating properties of NHC-ligands was exploited to make a hexa-carbene [Co(III)(phtmeimb)2]+ complex with microsecond excited state lifetime and 690 nm emission from a 3MC excited state (Fig. 52). This provides openings for photochemical applications via energy-transfer-driven singlet oxygen activation.913 

Other examples of first row transition metal complexes that show promising excited state properties for photochemical applications include several Cr(III) d3 complexes with long-lived excited states and near-IR emission,914,915 as well as Ni(II) d8 complexes with photochemical applications discussed further below.916 

4. Supramolecular systems

Supramolecular systems provide a modular and flexible approach to separate the light-harvesting by a dedicated chromophore and the catalytic activity that allows for separate optimization of the different molecular functions.917 A typical approach for constructing photoactive supramolecular systems is to link an efficient light-harvesting unit to an established redox-catalyst which is activated by photoinduced ET across the bridging unit.888,890,918,919 The photoinduced forward ET needs to be sufficiently efficient in terms of energetic driving force and rate to outcompete internal deactivation of the photosensitizer. At the same time the recombination needs to be sufficiently inefficient to suppress losses from recombination within the supramolecular system under operating conditions, where the dark electrocatalytic reaction can be much slower if it is governed by bimolecular diffusion. Measuring and controlling the forward and backward ET rates is thus central to mastering the functionality of supramolecular light energy conversion. A lot of work has been conducted on fundamental properties of such ET and related excitation energy transfer processes in a wide range of model donor-bridge-acceptor systems. In particular, the coupling of donor and acceptor units depends on both the length and chemical nature of any bridge that can act either as a tunneling barrier or a conduit of electron or energy transfer, as discussed in recent reviews.920–922 

Ru(II)-Co(III) heterobimetallic complexes have served extensively as model systems for activation of prototype Co-based reduction catalysts that can be efficiently activated by photoinduced electron transfer from Ru(II) polypyridyl photosensitizers.625 A combination of time-resolved optical and x-ray studies of the [(bpy)21RuII(tpphz)1CoIII(bpy)2]5+ [with bpy = bipyridine, tpphz = tetrapyrido (3,2-a:2′,3′-c:3′′,2′′-h::2′′′,3′′′-j) phenazine] system revealed key steps of the ET pathway (Fig. 53).923 This involved stepwise charge transfer via the phenanzine bridge between the Ru and Co centers on a sub-ps timescale. Subsequent structural stabilization of the reduced [Co(II)] center together with solvent reorganization resulted in the ultimate stabilization of the long-lived photoredox-activated Ru(III)-Co(II) state on a 1–15 ps timescale (Fig. 53).

a. Charge accumulation

Net transfer of multiple charges is essential for photocatalytic reactions that involve making and breaking of one or more chemical bonds and related rearrangements of electron pairs, with the classic example in this field being the need to rearrange a total of four electrons to accomplish one molecularly complete water splitting reaction (see Sec. IV F 1). This goes beyond the simple one-electron donation/accepting capabilities of many traditional one-electron photoredox species such as traditional Ru-polypyridyl photosensitizers. The interest in driving multi-electron chemical reactions has consequently prompted significant interest in molecular systems capable of charge accumulation as a stepping stone toward utilization in complex conversion reactions.625,888,924–926

A significant body of early work focused on the elucidation of the mechanistic photoinduced charge accumulation steps in biomimetic model systems, designed to allow for multi-electron storage. This included both stepwise oxidation of oligonuclear Mn clusters designed to mimic the water oxidation site in PS II and the stepwise reduction of oligonuclear Fe clusters designed to mimic hydrogenase enzymes.888 Three photoinduced oxidation steps of a Mn-dimer coupled to a Ru(II) photosensitizer could, for example, be demonstrated from a combination of sequential flashes with EPR monitoring of the sequential oxidation steps of the Mn2-complex from a Mn(II)-Mn(II) state to a Mn(III)-Mn(IV) over a narrow range of redox potentials.888 

Significant further progress has been made in recent years to develop various functional systems that utilize photoinduced multi-electron transfer (MultiET) charge accumulation to drive catalytic processes. Figure 54 shows a recent proof-of-principle example of a photocatalytic system comprising a dibenzo[1,2]dithiin catalytic center sandwiched between two Ru photosensitizers capable of driving two-electron organic reduction of DTT (trans-4,5-dihydroxy-1,2-dithiane) in conjunction with regeneration of the two photosensitizers by sacrificial electron donors.927 

Schulz et al. recently demonstrated a prolonged multielectron storage capable of separating the light and dark reactions in a single Cu-complex (Fig. 54 bottom panel). This allowed separation of the fast light-driven two-electron reduction of the Cu-complex from the dark reactions by up to 14 h, owing to the exceptional longevity of the catalytically active reduced species. This is in sharp contrast to many traditional D-A systems that often undergo recombination on a submillisecond timescale, at best, unless the recombination is interrupted by external regeneration, for example, from sacrificial electron donors/acceptors.928 

b. Proton-coupled electron transfer (PCET)

Proton-coupled electron transfer (PCET) reactions play an important role in several areas of photocatalysis. First, the connection between proton reduction and ET is of course directly relevant for the numerous proton reduction reactions that constitute one of the two main reactions in water splitting, as well as a broader range of conceptually related hydrogen atom transfer reactions. For example, the photo-driven reduction of several Co(III) catalysts to proton reducing Co(I) states using a photosensitizer have been investigated in significant mechanistic detail to identify at