Lasing at 602-620 nm from a red algae-derived phycobiliprotein

Derived from red or blue-green algae, phycobiliproteins are fluorescent protein pigments that continue to have a profound impact on medicine, pharmaceuticals, and food science.¹ Following the report of phycobiliproteins as bright fluorescence reporters in 1982 by Oi et al.,² applications developed rapidly and now range from photodynamic therapy to natural food colorants.³ The unique optical characteristics of phycobiliproteins, including quantum yields above 80% and the breadth of their absorption spectra, are critical to the survival of marine algae at depths beyond 30-70 m where photon fluences are low.⁴ In red algae, the light-harvesting complex comprises phycobiliproteins whose chromophores are known as bilins.⁵ Phycoerythrins are the most prevalent phycobiliproteins in red algae, and R-phycoerythrins (RPEs), specifically, are known to require 34 bilins,⁶ one of which is represented by the chemical structure of the upper inset to Fig. 1. The molecular weights of phycobiliproteins range from 100 to 240 kDa and, when extracted from red algae, fluoresce brightly in solution, and exhibit extinction coefficients ≤2.4 × 10⁶ M⁻¹ cm⁻¹ because of the localization of the bilins in the biomolecule.^5,7,8 Perhaps the most remarkable property of R-phycoerythrin is its fluorescence yield which is the equivalent, on a molar basis, of at least 30 fluorescein molecules.⁶ Considering also its biocompatibility, Stokes shift (>80 nm),⁷ and water solubility, RPE is clearly attractive for biophotonic applications. However, although RPE has proven effective as (for example) a fluorescent marker for biomaging, its potential as a biomolecular coherent source has not been realized previously. Indeed, lasing from phycoerythrins has long been presumed to not be possible.⁹ Although each R-phycoerythrin molecule, for example, is equipped with >30 chromophores, singlet-triplet conversion and energy transfer between neighboring bilins were expected to thwart the generation of coherent emission.⁹ 

We report here the demonstration of lasing in the 602-620 nm wavelength region from R-phycoerythrin extracted from red algae. When pumped by a frequency-doubled, Q-switched Nd:YAG laser (λ_p = 532 nm), solutions of RPE in a Fabry-Pérot resonator generate energies >26 nJ in 2.5 ns (FWHM) pulses. The threshold pump energy fluence is measured to be 260 ± 15 μJ/mm² for 8 μM solutions, and the cavity Q is 3.8 × 10⁶. Because the quantum yield and absorption spectrum breadth for RPE are superior to those of other recombinant fluorescent proteins, the RPE laser appears to be preferable to previous biomolecular lasers^10–13 when considering applications such as in situ laser imaging and polarization spectroscopy.

R-phycoerythrin was obtained from Thermo Fischer Scientific at a stock concentration of 4 mg/mL and was diluted to 8 μM with milliQ water. Because of the number of chromophores bonded to each RPE molecule, the density of the bilins in R-phycoerythrin is equivalent to a concentration⁶ of ∼80 mM. For these experiments, the optical cavity is a Fabry-Pérot arrangement comprising two flat mirrors with reflectivity R > 99% over the 600-700 nm wavelength interval. The transmission of both mirrors at the pump wavelength (λ_p = 532 nm) is >95% at normal incidence. A 10 μL droplet of RPE was first dispensed onto the surface of one mirror, and the second (top) mirror was then lowered by a linear, motorized translation stage until contact was made with the solution. Alignment of the Fabry-Pérot resonator was facilitated by interferometry with a He–Ne laser operating at 543 nm.

The frequency-doubled output of a pulsed Nd:YAG laser (8 ns, 10 Hz) was expanded by a spherical concave lens (f = −50 mm) and then directed into an Olympus 4× microscope objective by a dielectric mirror. Encompassed by the plane-plane optical resonator, the RPE solution was positioned at the working distance of the objective, and the diameter of the green pump beam at the gain medium was 0.7 mm. The resulting laser or spontaneous emission was collected by a tube lens and imaged onto the entrance slit (10 μm width) of a 0.75 m Czerny-Turner spectrograph operated in first order and having an intensified CCD detector at the exit plane. A 532 nm notch (bandstop) filter was inserted into the signal beam line to reject scattered pump radiation. Measurements of the energy and temporal history of the RPE laser pulses were provided, respectively, by a calibrated silicon detector and a photodiode having a <1.5 ns rise time. The lower inset of Fig. 1 is a simplified diagram of the laser resonator, associated optics, and pump beam.

Figure 1 summarizes the data acquired to date of the RPE laser pulse energy E_o, recorded as the pump pulse energy fluence Φ_p was varied up to 442 μJ/mm². Owing to singlet-triplet conversion and slight variations in the position of the pump focus, scatter in the data of Fig. 1 for a fixed value of Φ_p is evident. In order to estimate the threshold pump fluence, the linear, least-squares fit to the data was calculated and is represented by the dark blue lines of Fig. 1. The light blue-shaded region in the figure indicates the extent of the calculated 95% confidence interval. On the basis of this analysis, the threshold pump fluence of the RPE laser is 260 μJ/mm² when λ_p = 532 nm, and the uncertainty in this value is approximately ±15 μJ/mm².

Note from Fig. 1 that maximum single pulse energies >26 nJ have been observed which correspond to peak powers above 10 W for a 2.5 ns (FWHM) laser pulse (cf. Fig. 4, discussed below). Recalling that the pump beam diameter at the gain medium is 0.7 mm, the maximum pump energy delivered to the RPE solution is ∼155 μJ and the overall efficiency of the laser of Fig. 1 is ∼10⁻⁴. Because of the limited available temporal width of the pump pulse, this efficiency should not be viewed as reflecting the potential of the laser. As discussed later, approximately 60% of the duration of the pump pulse is required for laser threshold to be reached from the noise (spontaneous emission background).

The ∼0.02 nm resolution of the 0.75 m spectrograph permits the observation of longitudinal laser modes, and Fig. 2 is a laser spectrum recorded for a single pump pulse when the pump energy fluence is 600 μJ/mm². The peak laser output occurs at ∼610 nm and 112 longitudinal modes are observed. The inter-mode separation of Δλ = 0.18 nm matches the resonator free spectral range (FSR ≡ λ²/2nL) for n = 1.33 (refractive index of water at 600 nm and 300 K) and a cavity mirror separation of L = 777 ± 1 μm. Lasing extends over a spectral range of 21 nm or more than 50% of the spontaneous emission bandwidth⁷ of 41 nm. A magnified view of the RPE laser spectrum in the 609-612 nm wavelength interval is presented by the inset of Fig. 2. Since TEM_mn modes are degenerate in a plane-parallel (Fabry-Pérot) resonator, spectral features associated with specific Gaussian transverse modes are not readily noticeable in Fig. 2. It is also evident in Fig. 2 that virtually no amplified spontaneous emission (ASE) is present, appearing only in the wavelength region of peak gain (∼610-611 nm).

In an effort to convey the degree of reproducibility of the experiments, Fig. 3 displays another RPE laser spectrum, acquired with a pump fluence of 400 μJ/mm². Not surprisingly, transient heating of the gain medium by the pump pulse is capable of altering the envelope of the laser spectrum by increasing absorptive losses, predominantly to the blue side of peak gain. In effect, the Stokes shift between the RPE absorption and gain spectra is reduced because heating extends the population distribution in the RPE singlet ground state (S_o) to more highly excited vibronic levels. Assuming Beer-Lambert absorption by the RPE solution, most of the pump pulse energy is absorbed by the gain medium solution when the RPE concentration is 8 μM, α = (1.5–2.5) × 10⁶ M⁻¹ cm⁻¹, and L ∼ 800 μm. Consequently, non-radiative relaxation of RPE molecules in the lowest triplet manifold results in heating of the liquid gain medium and a localized alteration of the refractive index. The same can be said regarding the fraction of the energy of every absorbed pump photon that is dissipated as heat through relaxation of the RPE S₁ manifold.

Owing to the 700 μm diameter focal spot of the pump radiation, the induced refractive index profile resembles a microlens of the same diameter. Therefore, a transient spherical mirror-planar mirror optical cavity is formed, and it is this pump-induced resonator that breaks the degeneracy between the longitudinal mode frequencies of the TEM_mn transverse modes. This effect is evident in RPE laser spectra such as that of the inset of Fig. 3, an expanded view of the 607.1-607.9 nm portion of the overall spectrum, where weak but well-resolved features lie between adjacent longitudinal modes (indicated by the vertical arrows).

Temporal characteristics of the RPE laser emission are depicted in Fig. 4, in which the pump and laser waveforms are shown in black and red, respectively. For a pump fluence of 242 μJ/mm², the onset of lasing occurs ∼5 ns following the arrival of a pump pulse. Such a time delay, necessary for the intracavity optical field intensity to reach threshold, implies that the small signal gain coefficient (averaged over the 5 ns delay) has a lower bound of ≈0.13 cm⁻¹. For an RPE solution concentration of 8 μM, therefore, the data of Fig. 4 suggest that the lower limit for the stimulated emission cross section of RPE is ∼3 × 10⁻¹⁷ cm² which, being an order of magnitude below corresponding values for most organic dyes,¹⁴ is undoubtedly partially responsible for the RPE laser not having been demonstrated previously.

The peak in the laser pulse of Fig. 4 is delayed by ∼2 ns with respect to the maximum pumping rate, and the temporal width of the RPE laser waveform is ∼2.5 ns (FWHM) or less than one-half that for the pump pulse. Temporal narrowing is one of the key properties of pulsed lasers, confirming that lasing in RPE has, indeed, been demonstrated. The temporal decay of the laser pulse is well-described by a single exponential Ae^(−t/τ₁), where A is a constant, and τ₁ is found to be 1.24 ns, a value which is more than three times larger than the cavity ring-down time [τ_p = 2nL/c(1-R), where L = 777 μm and R ≡ 0.99].

The polarization characteristics of the RPE laser pulse were examined by directing the optical radiation through a polarizing beam-splitter. Both the horizontal and vertical polarization components of each laser pulse were detected simultaneously by matched photodiodes, and representative results are shown in Fig. 5. It is clear from the waveforms that the RPE laser emission is almost completely polarized horizontally, which matches the pump polarization. Only a small fraction of the laser output is vertically polarized which suggests that the rotational correlation time for RPE is long compared to its radiative lifetime.¹⁵ Owing to the rotational relaxation of organic molecules in solution, the degree of anisotropy for a liquid gain medium, in response to a polarized pump, is dependent on the rotational correlation time for the emitting species, relative to the lifetime of the upper laser level(s). Consequently, the observed polarization of the RPE laser emission suggests a high degree of anisotropy in the gain medium which, in turn, indicates that the 5 ns time delay between the arrival of the pump pulse and the initiation of lasing in RPE is insufficient for the molecule to relax rotationally.

In summary, stimulated emission from R-phycoerythrin, a phycobiliprotein derived from marine algae, has been demonstrated. Lasing is observed over a bandwidth of 21 nm in the orange-red region of the visible spectrum (602-620 nm). More than 100 longitudinal modes have been resolved and, owing to slow rotational relaxation of the molecule in solution, the polarization of the RPE laser radiation matches that of the pump. Despite the expected deleterious influence of triplet conversion and bilin-bilin excitation transfer on efforts to develop an RPE laser, lasing has been achieved in this phycobiliprotein with pulsed optical excitation in a Fabry-Pérot resonator. It is quite possible that the pumping intensities available with a Q-switched Nd:YAG laser are necessary to overcome non-radiative losses in the RPE visible oscillator, and experiments with long pulse (>1 μs) or continuous wave (CW) optical pumps will be necessary to determine if the non-radiative loss channels cited earlier will block attempts to extend the pulse width of this laser. Nevertheless, the extraordinary bandwidth of this biomolecular laser, combined with the prevalence of phycoerythrins as fluorescence tags, suggests that this new protein laser source will be of value for a broad range of biophotonic applications, including in situ laser imaging and polarization-sensitive diagnostics.

The support of this work by the U.S. Air Force Office of Scientific Research (J. Luginsland and J. Marshall) under Grant No. FA9550-14-1-0002 is gratefully acknowledged.