As studies on life sciences progress toward the single-molecule level, new experiments have put forward more requirements for simultaneously displaying the mechanical properties and conformational changes of biomolecules. Optical tweezers and fluorescence microscopy have been combined to solve this problem. The combination of instruments forms a new generation of hybrid single-molecule technology that breaks through the limitations of traditional biochemical analysis. Powerful manipulation and fluorescence visualization have been widely used, and these techniques provide new possibilities for studying complex biochemical reactions at the single-molecule level. This paper explains the features of this combined technique, including the application characteristics of single-trap and dual-traps, the anti-bleaching method, and optical tweezers combined with epi-fluorescence, confocal fluorescence, total internal reflection fluorescence, and other fluorescence methods. Using typical experiments, we analyze technical solutions and explain the factors and principles that instrument designers should consider. This review aims to give an introduction to this novel fusion technology process and describe important biological results.

  • Many fluorescence methods including Epi, confocal, TIRF, etc. were showed how to combine with optical tweezers.

  • Sample fixing methods, system structures and design principles in combined optical tweezers and fluorescence setup are discussed in detail.

  • Anti-bleaching principles and examples are widely collected and discussed including spatial and time separation methods.

The rapid development of single-molecule technology has allowed researchers to study the microstructure, movement, conformational change, interaction, and life mechanism of biomolecules at the single-molecule or catalytic level and help solve complex biological problems.1 The motion of biomolecules is always accompanied by a change in conformation. Increasing research on life activities requires revelation its comprehensive characteristics at the single-molecule level. A single mechanical or conformational study cannot easily meet measurement demands, and the rules of biomolecules are impossible to fully revealed if the two aspects are separated. For example, in the study of the mechanism of kinesin moving along microtubules, measuring the motion stepping force and the dynamic position of binding sites is necessary.2 In studies on protein unfolding mechanics, tensile force and the specific unfolding sites should be measured.3 In studies on the DNA unwinding mechanism, the unwinding force and sites of enzyme action are closely related to each other.4 The demand for joint measurement of force and conformation in biomolecules has raised new development issues for single-molecule research, and highlighted the urgent need for comprehensive measurement methods.

Optical traps were developed in the 1980s, and Ashkin et al. was the first to apply this technology to life science.5,6 Optical tweezers possess unique technical characteristics, such as non-contact measurement, micro-manipulation, nanometer positioning resolution, and mechanical detection at the piconewton level, and are gradually playing an important role in single-molecule mechanics; this technology can be used, for example, in initial studies of cells, bacteria, and viruses to proteins, nucleic acids, and membranes. Indeed, optical tweezers have already become an indispensable tool in frontier disciplines, such as molecular biology and biophysics, by providing an important technical guarantee for researchers exploring complex life mechanisms. Optical tweezers started late in single-molecule methods, but their outstanding performance continues to attract researchers to continuously innovate technology and develop applications, related to these tools, thus indicating their excellent potential.7 The main test ability is mechanical properties and the structure of setup was designed based on the force detection. The conformational changes of structures are impossible to take into account when revealing the mechanical properties of biomolecules.8 Single-molecule dynamics essentially involves 3D conformational changes, and optical tweezers are insufficient to capture this complex motion. All motions in the direction of the applied force are projected onto a single axis, such motion structure is bound to lose multi-dimensional motion information.9 Optical tweezers are inadequate as a single mechanical measurement method, and further development of its detection capability in microscopic imaging, molecular structure detection, and conformational analysis is necessary.

Compared with optical trap, the fluorescence microscopy is more capable of revealing biomolecule interactions, conformational changes, and dynamic activities. Fluorescent technology has a long history and rich in variety. It uses fluorophores labeled on biomolecules to study the mechanism of life structures and explain the process of nucleic acids and proteins participate in life activities.10,11 The appearance of fluorescence resonance energy transfer (FRET) makes it possible to measure spatial variations between different domains or subunits of biomolecules, which can characterize molecular spacing changes at the nanoscale. It plays an important role in protein-protein, protein-nucleic and acid dynamic interactions.12 Then, the intervention of a variety of high-sensitivity, low-background noise fluorescence technologies make the biological application more in-depth, revealing more complex phenomenon such as diffusion and translocation. Among them, there are mainly epi-illumination fluorescence microscopy, laser confocal fluorescence microscopy, total internal reflection fluorescence microscopy (TIRF), stimulated emission depletion microscopy (STED), etc. But fluorescent methods cannot detect the mechanical properties of biomolecules when showing the labeled points, and cannot be used as a comprehensive means for simultaneous measurement of force and conformation.

Optical tweezers and fluorescent techniques have complementary advantages and allow the simultaneous measurement of force and conformation. The time correlation between mechanical and fluorescent signals can be achieved by the combined acquisition of motion and conformational information. For example, one can relate structural rearrangements occurring within molecules to their larger-scale motions to explore the mechanisms underlying conformational changes. Similarly, molecular binding events observed by fluorescence can be related to structural changes perturbed by a load to determine the coupling between mechanical and biochemical cycles.13 However, combining the two technical means is not simple work, and several difficulties limit their development. For example, the two method use light sources of different intensities, and the trap and fluorescent excitation lasers must be effectively separated to ensure noninterference. The combination of these technologies complicates light-induced fluorophore bleaching.14–16 The original system is relatively complex, and the association will bring about a high degree of complexity in system structure, control and signal processing. Ways to achieve combination without sacrificing the sensitivity of either method must be further developed.9 Over the last two decades, however, despite these technical challenges, the combination has been successfully accomplished since the first generation of Steven's design model. It has evolved into a high-performance, integrated and new generation single-molecule technology.17,18

In this review, we analyze the combination of optical tweezers and fluorescent systems from a technical perspective and explain their innovation and development. We also classify these methods according to their technical characteristics and extensively describe successful cases reported in recent years. The purpose of this review is to highlight the advantages and discuss the new technical trend of combined single-molecule methods.

Optical tweezers mainly include single-trap and dual-trap systems, which differ in the way of samples are fixed objects.8 Single-trap are useful for studying the interaction of molecular motors with membranes and microtubules, while dual traps are more suitable for studying DNA-protein interactions. This difference affects their combination with fluorescence. Multiple and array traps may also be combined with fluorescence, using basically identical technical ideas.19–21 

The system structure of single-trap involves relatively simple equipment, and its operation is generally easy. However, because one trap cannot completely limit the movement of samples, the bead-sample-surface fixation method is often used (Fig. 1A, B). One end of the target protein is connected to the beads by DNA handles, and the other end is connected to the surface of coated slides. The force generated by the biomolecules can be calculated by the position of the beads deviated from the trap or by using the trap to exert a certain force on the target. This method facilitates the measurement of force, motion, and the target outside the trap beam, thereby providing a good excitation illumination environment for fluorescence.18 The main research objects are myosin,22 actin-binding protein,23 and kinesin.24 In addition, the use of fluid motion to fix samples laterally is often used (Fig. 1C). One end of the DNA is clamped to the beads. The DNA is kept stable and laterally fixed in the fluid direction under a constant rate flow. The motion force of DNA can be obtained by calculating the parameters of the fluid. The protein behaviors convert conformational changes such as repair, recombination and translocation into the lateral movement of the beads in the trap. This method is commonly used to study DNA repair proteins25,26 and recombinant proteins.27,28

Fig. 1.

Sample fixed method in a single-trap system. (A) The target protein is attached to the bead and the surface of the slide coated with antibody by DNA handles. (B) Myosin moves stepwise along the microfilament, and one end of the spherical head is connected to the bead by DNA handles. (C) The repair enzyme works on the DNA and stretched in the direction of the fluid.

Fig. 1.

Sample fixed method in a single-trap system. (A) The target protein is attached to the bead and the surface of the slide coated with antibody by DNA handles. (B) Myosin moves stepwise along the microfilament, and one end of the spherical head is connected to the bead by DNA handles. (C) The repair enzyme works on the DNA and stretched in the direction of the fluid.

Close modal

The sample-surface fixed method cannot guarantee that the sample is in a relatively controllable position, which is undesirable for accuracy fluorescent methods and suitable for fluorescence microscopy with extended imaging depth tolerance. Epi-fluorescence is the most successful technology to be combined with single-trap.29–31 Other successful combined cases such as confocal32,33 and TIRF4 with optical tweezers. The implementation is inseparable from detailed sample design, preparation and online microfluidic control.

Fig. 2 shows the key structure of a single-trap with fluorescent microscope. Trap and fluorescent lasers use the same objective. A suit of dichroic mirrors and filters are used to distinguish light and ensure mutual noninterference. Direct adjustment of the laser power will cause loss of stability, and a power conditioner is added to the optical path, which is composed of a splitting prism, and a controllable half-wave plate, among others. The trap beam is usually movable, a controllable micro-mirror is mounted in the optical path to drive the beam with scanning motion, and its position is optically conjugate with the detector.

Fig. 2.

Key structure of a single-trap and fluorescence microscopy system.

Fig. 2.

Key structure of a single-trap and fluorescence microscopy system.

Close modal

The target protein is not at the same focus plane as the bead, and a focusing lens is usually required in the fluorescent microscope to adjust the fluorescent irradiation position and maximize the excitation efficiency. Two cameras are usually used together: one is used to observe the position of the optical trap via a bright field microscopic method, and the other is used as a detector for fluorescence signals. In this combination mode, the optical trap can be controlled to apply force or fix the optical trap to detect molecular motion. Then, the fluorescent labeling site on the target molecule emits fluorescence under the excitation laser (Fig. 2). The emission light is collected into the camera through the objective to detect conformational signals.

Over the past 20 years, this combined technology has made outstanding contributions in research on the motion mechanics of motor proteins and the relationship between mechanical motion and ATP hydrolysis. When the labeled ATP molecule binds to the protein, a fluorescent spot appears; if ATP is hydrolyzed to produce ADP, the fluorescence disappears.34 Using this hydrolysis process and microfluidic technology,35,36 researchers have extensively revealed the intrinsic link between the stepping motion and energy supply of myosin-IV and myosin-V along microtubules.22,37,38 This technique has been applied in studies of mitosis and meiosis to examine the mechanism of motor proteins attached to microtubules to drive chromosome segregation. Green fluorescent protein was used as a connected marker for kinetochores and beads, microtubule structures were attached to the surface of the sample cells, and the binding force and action times of the kinetochore and microtubule chromosomes were analyzed by optical force quantification.39–41 Lee et al. fixed the ssDNA with dsDNA by the same method, and studied the binding force of fluorescently labeled Escherichia coli helicase UvrD with DNA.4 Using fluid fixation, Bianco et al. accurately measured the rate of uncoupling base pairs of RecBCD in the study of homologous recombination-related enzymes.25 

The dual-trap system splits an additional optical trap from the laser source based on the single-trap system. The two beams are simultaneously concentrated in the sample cell to form two optical traps through the objective. The dual-trap system is a powerful tool for dynamic process detection and interaction analysis of biomolecules. Compared with the single-trap system, the sample is totally controlled by laser and the freedom of motion is strictly limited, and the accuracy and stability of force measurement is further ensured. Dual-trap system has a more complex instrument structure, precise mechanical exploration capabilities, and motion control capabilities.42 Many studies focusing on the design and construction of dual-trap devices have been reported, and the current design can achieve very high stability and accuracy than single-trap system.43,44 The dual-trap system has excellent applications, and it can accurately reflect piconewton-level forces and the nanometer position in millisecond-scale time resolution.3 

The two traps can be positioned in the horizontal direction without surface fixation of the samples in the dual-trap configuration, thus ensuring that target molecules are manipulated in an approximately horizontal direction and reducing the uncertain mechanical component and the risk of surface interference. The sample is horizontally fixed by DNA handles between the two beads, and the lateral force can be sensitively measured by optical traps through light-intensity detectors (Fig. 3). A fluorescent laser (green) illuminates the sample area between the two trap beams (red) to excite labeled sites (yellow).45,46 Such horizontal operation also facilitates fluorescence imaging positioning. Epi-illumination fluorescence evenly illuminates the entire sample plane area, and other common fluorescence techniques can be used. The confocal fluorescence structure is the best and most frequently used technology in combination with dual-trap systems.47,48 TIRF, STED, and other fluorescence methods were also successfully combined.49–51 

Fig. 3.

Sample test model under a dual-trap and fluorescent system.

Fig. 3.

Sample test model under a dual-trap and fluorescent system.

Close modal

A fixed-position trap and a controllable trap are employed in a typical dual-trap structure. The controllable trap is accurately positioned on the two sides of the fixed-position trap.45 The key structure of the dual-trap system is basically identical to that of the single-trap, especially in terms of the objective lens and sample area. The technical means of optical integration with fluorescence is equally applicable. The trap laser, fluorescent excitation, emission light, and illumination light in the system are distinguished by using a set of dichroic mirrors and filters. The center of the optical trap and the sample are in the same horizontal plane under ideal conditions. Some deviation may be expected due to system installation and device errors,52 and the focus lens that adjusts the fluorescent excitation position cannot be omitted. After the optical system is set up and debugged, the axial position of the trap is fixed. Thus, the axial position of the sample cannot be adjusted. The focus lens can also assist the beam to accurately illuminate the fluorescent site.

Dual-trap fixation mode can accurately bind biomolecules between the two beads. Lateral motion allows convenient dynamic fluorescence analysis. It is widely used in DNA mechanics, helicase, DNA intercalating dyes, membrane fusion, bacterial movement, and molecular motion.18 DNA has fluorescent binding sites or intercalators.53 DNA stretching and their combination or dissociation will change the length of DNA chains and affect their fluorescence intensity.54,55 The fluorescent beam can excite all relevant sites on the entire chain to realize simultaneous detection of multiple points.56 Another studying method is to label DNA on related enzymes, such as RAD51.57 Enzymes with dyes in the sample solution bind to DNA, and the DNA length changes because of enzyme activity, such as repair and unwinding.58 This system can also be used to study membrane fusion. With the help of Doc2b and SNARE receptor,59 the membrane on the surface of beads can be fused, and the labeled lipids diffuse to another bead. The fluorescence intensity gradually increases during fusion, and the trap can detect the fusion force.60 In the study of bacterial movement, two traps were separately fixed at the head and tail of E. coli to restrict its space movement but not rotational motion.61,62 Fluorescence imaging processing techniques are then used to detect its periodic tumble rotation rate. In this research method, the optical tweezers were used to limit motion freedom, while fluorescence detection was applied for motion analysis. Thus, the extensiveness and flexibility of this combined instrument was fully demonstrated.

The dissociation motion of biomolecules or photobleaching leads to attenuation of fluorescent signals. The typical trap power is at the watt level, and the fluorescent excitation power at the focusing point is about 100 mw/cm2. Typical fluorophores produce about 104–105 photon fluxes per second and are femtowatt level compared with the excitation light.63 Distinguishing weak fluorescent signals and the bright light of the excitation background is important in combined technology and determines fusion performance. The essential elements of technology integration include reasonable samples, fluorescent dye selection, laser power control, filter performance, and data processing methods.

During exploration of factors affecting the stability of fluorophores, van Dijk et al. focused on the bleaching of dyes under the influence of the trap and fluorescent excitation lasers.16 This experiment used an 850 nm trap laser and a 532 nm fluorescent laser. The bleaching effect of the 25 mW trap power on the fluorescence intensity of the Cy3 molecule was exponentially fitted with time, and the decay constant was 0.78 (Fig. 4A, B). This study proved that the bleaching rate increases linearly with increasing intensity of both lasers. The observed enhancement of bleaching is caused by electrons in the excited state continuing to absorb near-infrared photons. The experiment also verified the bleaching tolerance of three dyes (Fig. 4C). Tetramethylrhodamine (TMR) showed the best resistance to enhanced bleaching, and addition of an antioxidant to the solution relieved bleaching. Ishijima et al. studied the probability of photobleaching49 and showed that the photobleaching rate of Cy3-labeled nucleotides bound to actin is about 1000 photons per second. Trap experiments have proven that the binding rate constant of actin is approximately 0.13 s−1, which is far greater than the photobleaching rate. Therefore, 94% of the fluorescence decay is caused by the dissociation of Cy3-labeled nucleotides and the remaining 6% is due to bleaching that occurs before dissociation. Replacing traditional fluorescent dyes with new enhanced-intensity dyes appears to be feasible. Handa et al. introduced strong fluorescent nanospheres as markers.26 Despite their enhanced fluorescence, however, the cumbersome dye ball added more uncertainty to measurements and sacrificed the lightness and flexibility of molecular dyes. Biebricher et al. used high-brightness and stability fluorescent quantum dots as markers64 and accurately tracked the activity of the EcoRV enzyme on DNA. However, the effect of quantum dots on the biochemical activity of enzymes and DNA is unclear and may lead to bias in the measurement data.

Fig. 4.

Effect of optical power on photobleaching (adapted from van Dijk et al.16). (A) Dependence of the trap power on photobleaching. The fitting line indicates that the average bleaching rate of Cy3 on the surface of 10 beads increases linearly with the trap power. (B) The average bleaching rate of Cy3 on the surface of 10 beads increases linearly with the excitation intensity. (C) Relationship between bleaching rate of Cy3, Alexa, TMR dyes, and trap power.

Fig. 4.

Effect of optical power on photobleaching (adapted from van Dijk et al.16). (A) Dependence of the trap power on photobleaching. The fitting line indicates that the average bleaching rate of Cy3 on the surface of 10 beads increases linearly with the trap power. (B) The average bleaching rate of Cy3 on the surface of 10 beads increases linearly with the excitation intensity. (C) Relationship between bleaching rate of Cy3, Alexa, TMR dyes, and trap power.

Close modal

In terms of system design, the instrument must be equipped with high spectroscopic efficiency dichroic mirrors and filters with excellent passband performance. The emission light should be filtered before entering the detection camera. The trap laser has a typical wavelength of 1064 nm and the range of excited and emitted light is usually 450–700 nm. Thus, 800 nm near-infrared light or 400 nm blue LED light can be used to fit the dichroic mirror and filters. The wavelength response of the fluorescent camera can also have a certain inhibitory effect on the background light. The visible light-receiving camera can reduce the influence of the infrared background. The trap and fluorescent modules should be packaged and shaded to minimize scattered and ambient light interference.

In terms of data acquisition, real-time acquisition and correlation between motion and fluorescent signals is an important indicator for evaluating the performance of a combined instrument. The motion signal is obtained by analyzing the position of the beads, and the trap system usually uses the back focal plane interferometry (BFP) method to measure motion in real-time. BFP is based on a light position-sensitive detector, such as a four-quadrant detector, which can provide millisecond-level time responses. Fluorescence signals are obtained by light plane array detectors, such as CCD and CMOS cameras. The acquisition and time resolution of various cameras are quite different. If the subject has a short molecular motion time, a high-speed camera is needed to meet measurement requirements; a high frame rate acquisition mode leads to insufficient exposure time and is unsuitable for capturing weak fluorescent signals. The acquisition and processing rate differences of these two types of detectors leads to the loss of some data information. Focusing on the key signals at specific moments can reduce the processing pressure of fluorescent signals. The signals of motion and fluorescence strictly correspond in timing. High-speed data acquisition equipment must be used to ensure the speed and stability of data from transmission to processing. The upper computer should adopt sufficient automation and low time-consuming processing procedures.

According to Komori et al., the effect of the trap laser on the photobleaching rate of Cy3-labeled nucleotides can be described by a Gaussian distribution function (unpublished), so the trap laser has no effect on the fluorophore at 4 μm (>3σ) away. Increasing the DNA handle can reduce the photobleaching probability and is the simplest and most straightforward method to address this issue. The two beams are separated at a spatial distance longer than 4 μm so that they do not affect each other. This technique does not improve the instrument layout, but problems such as unstable traction, inaccurate force measurement, and easy breakage of the connection point limit its application.

In 1998, Ishijima et al. set two optical traps at a distance of 15 μm apart with a microtubule filament in the middle to detect single actin and ATP motion; the trap laser had a very weak effect on the Cy3.49 Harada et al. expanded the DNA handle length to nearly 50,000 base pairs (approximately 16 μm) in the interaction between RNA polymerase and DNA.65 Hohng et al. separated the trap from the labeled position at about 13 μm in the Holliday study of the recombination mechanism.66 Such lengths of DNA handles are extremely challenging for optical devices and samples. Long-range, controllable large-angle mirrors are required and the stability of the attachment is greatly reduced. A longer DNA means the beads and DNA-binding sites are under more pulling strength, thereby directly affecting the success rate of DNA and beads connecting during the experiment proper. DNA molecules have a certain degree of scalability due to their helical structure.67 The longer the DNA chain, the larger the expansion length, the slower the time the force is transmitted to the beads, and the larger the cumulative error.

The spatial separation method can significantly reduce photobleaching, prolong the lifetime of fluorophores, and increase the observation time at low technical cost. However, the trap laser and fluorescence are always in the illumination state, and the emission light is affected by the background noise from the trap laser. This method is limited in the observation of lower fluorescence intensity groups and cannot provide a low-noise fluorescence observation environment. Moreover, it cannot be used in some short-strand DNA length change studies with DNA lengths less than 1 μm.68 

Another way to solve photobleaching is to force the trap laser and fluorescence to shift over time. Only one laser is irradiated on the sample area at the same time. The two beams are separated in time so that they always act on the fluorescent dye separately. Brau et al. pioneered this proposed solution to increase the photobleaching lifetime of Cy3 by 20 times compared to fluorescence and trap laser simultaneously in 2006.69 

The time separation method needs carefully select the frequency of the switching light sources. The trap laser continuously illuminates the bead to provide the binding momentum. The laser must be switched at a very high frequency to make the beads receive continuous illumination over short time intervals so that the traps function normally and do not to fail. However, the optical system has extremely low tolerance for additional vibration and noises, and mechanical modulation switches are not feasible. High-frequency crystal laser modulators, such as acousto-optic modulator (AOM) and acousto-optic deflector (AOD), have been introduced to achieve this technology. These diffraction devices can modulate beams over a bandwidth of up to 100 MHz. Two identical modulators are placed in the fluorescent and trap optical path. The RF signal generator excites and emits two square wave pulses with the same timing and opposite levels to modulate the two lasers out of phase.

Study of time-sharing traps have revealed that the trap stiffness is determined by the modulation frequency and duty cycle.70 Experiments have confirmed that the positional shake of the beads generated by the modulated beam in the frequency range of 100 Hz to 10 kHz is significantly reduced (Fig. 5A) and the trap stiffness is increased. At 10 kHz, the upper limit is reached and no clear change can be observed (Fig. 5B). At a constant laser power, when the modulation frequency is five times the characteristic frequency of the system, the modulated trap stiffness is about 99% of the continuously generated trap, and the modulation frequency cannot be less than 10 kHz. Dyes exposed to intermittent excitation light can be considered to present sustained emission due to the presence of a fluorescence lifetime.

Fig. 5.

Time separation method. (A) Beads position shakes at 100 Hz, 1 kHz and 10 kHz modulation frequency of trap beam (adapted from Brau et al.69). (B) Relationship between the trap stiffness of four laser powers and modulation frequency (adapted from Brau et al.69). (C) Chronological logic diagram showing modulation of the fluorescence and trap beam by 30% and 50% of the duty cycle. The 10% period between two fluorescence open portions is idle. The histogram indicates the photobleaching condition of Cy3 after alternate modulation (adapted from Brau et al.69). (D) Modulation of fluorescent beam (green) and trap beam (orange) by a 50% duty cycle a (adapted from Sirinakis et al.71). (E) The trap AOM driving signal (orange) alternately forms two optical traps at different levels in the first 2/3 cycles, and the fluorescent AOM driving signal (green) is only switched on in the last 1/3 cycle (adapted from Comstock et al.68).

Fig. 5.

Time separation method. (A) Beads position shakes at 100 Hz, 1 kHz and 10 kHz modulation frequency of trap beam (adapted from Brau et al.69). (B) Relationship between the trap stiffness of four laser powers and modulation frequency (adapted from Brau et al.69). (C) Chronological logic diagram showing modulation of the fluorescence and trap beam by 30% and 50% of the duty cycle. The 10% period between two fluorescence open portions is idle. The histogram indicates the photobleaching condition of Cy3 after alternate modulation (adapted from Brau et al.69). (D) Modulation of fluorescent beam (green) and trap beam (orange) by a 50% duty cycle a (adapted from Sirinakis et al.71). (E) The trap AOM driving signal (orange) alternately forms two optical traps at different levels in the first 2/3 cycles, and the fluorescent AOM driving signal (green) is only switched on in the last 1/3 cycle (adapted from Comstock et al.68).

Close modal

Means to control the cycle between two beams alternating in a cycle have been developed to minimize photobleaching and maintain a certain trap stiffness, and several research groups have given different empirical solutions. Brau et al., for example, set the average power of the fluorescent and the trap laser to 100 mW and 250 mW, 30% and 50% duty cycle at 50 kHz, respectively (Fig. 5C). The fluorescence and trap laser durations are 10 and 6 μs, respectively, and a 2 μs dark period is set between adjacent pulses.69 Sirinakis et al. set the AOD with a 27 MHz sinusoidal excitation signal in the trap path and the AOM with an 80 MHz sinusoidal excitation signal in fluorescent path; each signal also had a 100 kHz frequency and 50% duty cycle (Fig. 5D). They maintained spatial resolution of single base pairs and fluorescent time of nearly 90 s.71 Comstock et al. adopted a special trap control method.68 The AOM in the trap path not only provided switching but also deflected the trap at two angles with high frequency through different levels of control (Fig. 5E). Instead of forming and controlling two traps by using beam splitting prisms and micro-mirrors, the two traps were formed by AOM modulation. Therefore, in the alternating cycle arrangement, the frequency was modulated at 66 kHz, and the durations of the two optical traps were each 1/3 cycled. The last 1/3 cycle closes the two traps and switches on the fluorescent laser. The fluorescence detector was only turned on in the last cycle and collected the emission data. The performance of this method was verified by the hybridization process of Cy3-labeled ssDNA and dsDNA.9 Suksombat et al. applied the same 66 kHz modulation setup to study dsDNA binding protein All non-standard abbreviations, acronyms, and symbols used in the Abstract and text must be spelled out at first or lone mention.72 

The time separation method successfully solves the problem of photobleaching without extending the DNA handle and does not require special fluorescent dyes to significantly increase the fluorescent lifetime. However, the implementation of time separation is much more complicated. For example, the adjustment angle of the acousto-optic diffraction device needs to be accurately positioned, and hardware requirements are needed including the control trigger of the RF signal, the detector control and the degree of instrument automation. Although experiments have shown that the trap generated by alternating switching has sufficient stiffness, the bead shakes generated by the periodic variation are likely to introduce noise to the position detector at the sub-millisecond level. This noise band is close to the position detector, filters are difficult to eliminate. Despite certain technical difficulties and measurement problems, however, the time separation method remains the best way to solve photobleaching. Some research groups have followed this design and developed approaches to improve the processing speed, measurement accuracy, and noise reduction of the device.

Epi-illumination fluorescence is the most basic fluorescence imaging technology. The imaging field is wide and the fluorescence intensity is strong, but the fluorescence depth resolution is limited. Moreover, the final imaging is affected by large background noise. Long-term exposure is always employed to enhance the imaging signal-to-noise ratio, resulting in poor time resolution. Scanning, similar to that in confocal microscopy, is unnecessary; thus, the technique is suitable for the detection of large spaces and samples with strong fluorescent signals. Due to its simplicity and ease of implementation, epi-illumination fluorescence has become a pioneer technology in combination with trap systems and has rich applications in single- and dual-trap systems.73–75 

In the structure of an epi-illumination fluorescence microscope (Fig. 6A), the excitation laser is vertically irradiated onto the surface of the sample through the objective, and the stimulated emission light is collected into the camera without the scattered light, which is removed by the dichroic mirror and filters. The objective is used as both a concentrator for fluorescent excitation and a collector for fluorescence emission. Thus, the objective and concentrator in the imaging system are always strictly aligned, and the field of view is always the fluorescence excitation area. The full numerical aperture of the objective is available under well Kohler illumination conditions. Epi-illumination observation needs to be equipped with a plane array detection camera. If analysis of the change in movement and position of several labeled points is required, a high frame rate camera and image processing method should be used. The epi-fluorescence structure can be directly coupled to the trap system through a dichroic mirror and share the same objective with the optical trap laser.

Fig. 6.

Optical tweezers with epi-illumination fluorescence. (A) Major structures of an epi-illumination fluorescence microscope. (B) Fluorescent background noise contrast of ssDNA; left and right images represent the imaging of buffer channels containing fluorescent molecules and non-fluorescent buffer channels, respectively (adapted from Gross et al.63). (C) Snapshot continuous frames show folding and unfolding process in a single DNA molecule fixed between two beads (adapted from van den Broek et al.76). (D) Fusion processing of a fluorescein-containing lipid membrane. The fluorescence images are captured at three moments (adapted from Brouwer et al.60).

Fig. 6.

Optical tweezers with epi-illumination fluorescence. (A) Major structures of an epi-illumination fluorescence microscope. (B) Fluorescent background noise contrast of ssDNA; left and right images represent the imaging of buffer channels containing fluorescent molecules and non-fluorescent buffer channels, respectively (adapted from Gross et al.63). (C) Snapshot continuous frames show folding and unfolding process in a single DNA molecule fixed between two beads (adapted from van den Broek et al.76). (D) Fusion processing of a fluorescein-containing lipid membrane. The fluorescence images are captured at three moments (adapted from Brouwer et al.60).

Close modal

The surrounding area around the sample is inevitably irradiated by fluorescence when epi-illuminated. The marked molecules in the solution that do not bind to the test sites are also illuminated, resulting in a large background noise. To solve this problem, Noom et al. controlled separation by using a fine layered microfluidic structure.63,77 Here, the trap was manipulated to move the beads and sample into the channel containing the fluorescent molecules only a few seconds after the sample connected, and the excitation laser was switched on after the beads and the sample were moved back into the fluorescent buffer channel. This method greatly reduces the concentration of unbound fluorescent molecules in the test environment and background noise to highlight the main fluorescent information and improve image clarity and quality (Fig. 6B). The beads were observed in fluorescence images because fluorophores in the buffer adhere to their surface (Fig. 6C). This mechanism enables easy observation of the position of the beads in the camera and helps determine the labeled point.76,78 Omitting the trap camera by coating the surface of the beads with dyes is also possible.60 The beads and fluorescence site can be directly observed by the fluorescence camera (Fig. 6D).

Epi-fluorescence cannot meet the sensitivity requirement when the fluorescence intensity. The ability to detect weak fluorescent signals places new demands on fluorescence methods. Laser scanning confocal microscopy and optical tweezing technology were developed at approximately the same time; the former is a powerful microscopic method for high-resolution image lossless optical sectioning of biological cells or tissues. The application covers various fields of life science, including the dynamics of protein molecules, changes and tracking localization, membrane potential conduction, cell proliferation and differentiation, neurology, and pharmacology. Two-photon, multiphoton, micro-slice and other confocal microscopy techniques were since been developed.79 The confocal structure is suitable for various sample fixing methods and is not affected by the sample position. Many successful combination cases based on single- and dual-trap systems have been reported.47,80,81

The confocal technique uses a conventional fluorescent microscopic structure to form a point source through a pinhole. The illuminated point on the target is imaged at the probe pinhole and point-by-point received by a photodetector, such as a photomultiplier tube (PMT) or CCD. Confocal laser uses the scanning excitation beam to obtain the entire cross-sectional shape information. The pinhole shields the scattered light of the unfocused plane and outside the non-focused point on the focal plane by a spatial light filtering function, thus ensuring that all of the fluorescence signals received originate from the focus point. Such method significantly improves optical resolution and imaging quality.79 The confocal structure employs more pinholes and scanning parts compared with the epi-fluorescence layout, and the system complexity is improved. However, no significant difference in terms of the coupling method with optical tweezers exists between these two techniques.

A compact confocal microscope uses only microwatt-level excitation laser power, but the confocal system used on the trap system requires milliwatt-level power to compensate for optical path scattering and diffraction device consumption. The trap system is suitable for use with the non-scanning confocal structure (Fig. 7). Expanding the fluorescent incident beam to the aperture of the objective lens reduces the alignment difficulty of the trap and fluorescent beam. Lens1 and lens2 not only function as beam expanders but also allow axial translation to enable fine adjustment of the focal depth of the excitation beam. The moving mirror is used to adjust the position of the fluorescent beam on the focal plane to maximize the excitation. The emission light is processed by the dichroic mirror and the filter and then enters the pinhole after filtering out of non-focus scattered light. Whitley et al. gave the experience value of the pinhole size is about 20–100 μm.9,82 Two lenses are fixed in front of and behind the pinhole; the first lens focuses the emitted light into the pinhole, and the second lens collects the qualified transmitted light onto the detector. Fluorescence detectors should use point-sensitive type with high sensitivity photoelectric conversion and high-speed acquisition capability, such as avalanche diodes and PMTs. A typical scanning confocal system performs two-dimensional scanning on the focal plane of the sample to acquire tissue and cell sections. When combined with the trap system, the labeled sites are usually detected on the DNA handle in the middle of the two beads, and a one-dimensional line scan is adequate.72,83 Ensuring controllability in another plane dimension is necessary so that the fluorescent beam can be accurately positioned toward the central area. Cell or tissue confocal scanning can also be used to study the related characteristics.84Fig. 8A and B show a confocal fluorescence image with trap fixation.80 

Fig. 7.

Major structure of a dual-trap with confocal fluorescence system.

Fig. 7.

Major structure of a dual-trap with confocal fluorescence system.

Close modal
Fig. 8.

Optical tweezers with confocal fluorescence. (A) Beads fixed by traps are connected to HEK293 cells and labeled F-actin under a confocal microscope, visible short filopodia on the surface of the cell (adapted from Leijnse et al.80). (B) Deconvoluted 3D reconstructed images of fluorescent-labeled F-actin filaments (adapted from Leijnse et al.80). (C) A single-trap protocol for measuring SSB protein-bound ssDNA by FRET. The curve shows the force-fluorescence relationship obtained by stretching and relaxing DNA and the change in fluorescence intensity for the donor and receptor (adapted from Zhou et al.33). (D) Donor and acceptor fluorophore positions of UvrD for FRET measurement. Conformational transformation experimental models for UvrD involved DNA repair. The color image represents the correlation between UvrD activity and conformation and the distribution correlation between average FRET efficiency and UvrD motion velocity (adapted from Comstock et al.47).

Fig. 8.

Optical tweezers with confocal fluorescence. (A) Beads fixed by traps are connected to HEK293 cells and labeled F-actin under a confocal microscope, visible short filopodia on the surface of the cell (adapted from Leijnse et al.80). (B) Deconvoluted 3D reconstructed images of fluorescent-labeled F-actin filaments (adapted from Leijnse et al.80). (C) A single-trap protocol for measuring SSB protein-bound ssDNA by FRET. The curve shows the force-fluorescence relationship obtained by stretching and relaxing DNA and the change in fluorescence intensity for the donor and receptor (adapted from Zhou et al.33). (D) Donor and acceptor fluorophore positions of UvrD for FRET measurement. Conformational transformation experimental models for UvrD involved DNA repair. The color image represents the correlation between UvrD activity and conformation and the distribution correlation between average FRET efficiency and UvrD motion velocity (adapted from Comstock et al.47).

Close modal

Confocal fluorescence microscopes have sufficient detection sensitivity and are suitable for serving FRET, allowing a very small-scale conformational change detection in nanometer or sub-nanometer level. The FRET consists of a fluorescent pair, i.e., a donor and an acceptor, and their emission wavelengths differ by about 50–100 nm. When the spatial distance between the donor and acceptor is less than 10 nm, fluorescence energy transfer occurs. The donor fluorescence intensity decreases, while the receptor increases, and the transfer intensity increases with decreasing distance between the donors. Fluorescence detection components need a two-color detection structure based on the monochrome structure because the emission light has two bands; thus, filters and an additional detector are added behind the pinhole.47 FRET technology essentially allows ratio measurement wherein the transfer efficiency is calculated by the ratio of the luminescence intensity of the acceptor. It is not susceptible to noise, but the trap laser has a slightly different effect on the two dyes at infrared wavelength. The emission signal is extremely weak and has a short duration. Thus, the time separation method is recommended to ensure anti-bleaching ability.

Confocal FRET is widely used with single- or dual-trap systems. Zhou et al. studied the effects of SSB protein dissociation from ssDNA, DNA tension, and protein interactions on the process in a combined single-trap and confocal fluorescence device (Fig. 8C).33 Comstock et al. studied the function and structure of the DNA repair enzyme UvrD in a combined dual trap and confocal fluorescence layout (Fig. 8D).47 Debjit et al. studied fluorescence changes in the dye attached to the surface of beads in traps under the femtosecond FRET probe laser and then analyzed the related molecular structure and kinetics.85 The combination of optical tweezers and FRET technology yields an extremely fine measurement system that characterizes the mechanical properties of single-molecule activity at the piconewton and sub-nanometer conformations, thereby reflecting its extremely accurate measurement capability.

Total internal reflection fluorescence microscopy (TIRF) can be combined with optical tweezers. In TIRF, the excitation laser emits a critical total reflection on the surface of the sample chamber to produce an evanescent wave that is exponentially attenuated; here, the evanescent wave excitation region is located 100–200 nm close to the surface. Background noise and Rayleigh scattering interference from most samples are reduced. Sample solutions beyond the excitation region are not excited, thereby reducing the fluorescence loss. This design has been widely used in single-molecule fluorescence imaging and FRET.86 The excitation laser of TIRF is incident at a critical angle rather than perpendicular to a surface, which is quite different from the Epi and confocal in combination with the trap system.

TIRF features two instrument structures: prism type and objective type. Although the objective type presents excellent features and the sample placement is flexible, the special objective used by TIRF is unsuitable for forming traps due to process limitations. The combined cases successfully reported are mostly prism type TIRF. The trap system is coupled into the TIRF microscope, and the trap laser and fluorescence collection use the same objective (Fig. 9). The condenser in the original trap system is replaced by a TRIF prism, and the incident angle of the excitation laser is adjusted by the moving mirror to form a critical total reflection state. Discarding the condenser means the transmissive trap position detection method cannot be used49 but is suitable for the reflective method and illumination light. The sample is oriented adjacent to the prism surface; here, using the geometry of a single-trap to place the sample attachment point on the surface is a good choice.87 Harada et al. applied this configuration to a dual-trap; the authors fixed prism on a micromechanical positional pedestal and adjusted it within 200 nm of the sample, thereby allowing evanescent waves to be accurately excited onto the enzyme binding site attached to the DNA handle.65 

Fig. 9.

Major structure of an objective type TIRF and dual-trap system.

Fig. 9.

Major structure of an objective type TIRF and dual-trap system.

Close modal

Later, there was an objective type TIRF combined with optical tweezers, Lang et al. used new objective (100×/1.4NA oil immersion).13 Such objective had good light transmittance and achromatic ability in the visible and infrared light bands. The objective-type TIRF can directly place evanescent wave fields in close proximity to the coverslip; it also showed good trap stiffness and could reduce the trap power required to capture and slow down photobleaching. This instrument mechanism only needs to incorporate the excitation laser with controllable incident angle into the objective through the dichroic mirror. The angle of incidence of the excitation laser can also be adjusted using a micrometer, and the position of the fiber aperture of the excitation light relative to the collimating lens is precisely adjusted.86 The condenser collects the trap laser to the position detector, and the position of beads can still use the BFP method. The emission light returns from the objective to the detector.88 

TIRF has good fluorescence imaging capabilities, and the combined layout further improves the measurement accuracy of filament-binding protein kinetochore-chromosomes and cortical microtubule attachments. Deng et al. measured sub-composites in a single kinetochore with an accuracy of 2 nm based on a single-trap and TIRF configuration.89 Ishijima et al. studied actin movement by using Cy3-labeled ATP, Cy5-labeled actin, and two types of excitation lasers. The group simultaneously observed the hydrolysis of ATP molecules and the movement of individual proteins using the two-color TIRF system.49 Lee et al. measured the relationship between the DNA repair helicase UvrD translocated on ssDNA and tension by using a long ssDNA stretched to an oblique angle near the surface in single-trap to retain it in the evanescent wave excitation field, thereby enabling tracking of the position of a single labeled UvrD over a longer distance.4 Lin et al. used the objective-type TIRF to study the relationship between the translocation rate of NS3h and the tensile force in ssDNA.90 

STED is a kind of super-resolution technology developed to allow far-field optical microscopes to break through the optical diffraction limit. STED makes improvements on the fluorescence excitation beam. An excitation beam is used to excite the target in the diffraction spot, and a circular lossy beam is used to illuminate the target coaxially. STED suppresses the fluorescence intensity around the labeled point, making only a dozen nanometers wide of fluorophores emitted and observed. STED is used in conjunction with a confocal microscopy layout. Heller et al. combined optical tweezers with a multi-color confocal and STED to observe individual DNA-binding proteins in the presence of densely packed DNA and high protein concentrations. The protein on DNA as visualized at 50 nm resolution, which is six times greater than that provided by traditional confocal microscopes (Fig. 10A). This system utilizes fast one-dimensional beam scanning to ensure a time resolution of less than 50 ms and could distinguish and track the trajectory of a protein translocated on DNA.51 

Fig. 10.

Other combined technology. (A) Confocal and STED imaging. The STED can clearly distinguish proteins in close proximity to dense areas compared with the confocal device (adapted from Heller et al.51). (B) The polarized fluorescence image of the DNA molecule continuously in the extension of the three length ratio and the corresponding tensile force. The intensity of the parallel fluorescence polarization component decreases with increasing tension (adapted from van Mameren et al.91). (C) The four traps control four beads, and the XRCC4-XLF protein bridge formed by incubation is connected in the middle. The protein at the node is clearly excited (adapted from Brouwer et al.19). (D), 3 × 3 array trap intensity image shows the relationship between fluorescence intensity and tPSA concentration including beads coated with a single antigen and mixed antigen (adapted from Cao et al.20).

Fig. 10.

Other combined technology. (A) Confocal and STED imaging. The STED can clearly distinguish proteins in close proximity to dense areas compared with the confocal device (adapted from Heller et al.51). (B) The polarized fluorescence image of the DNA molecule continuously in the extension of the three length ratio and the corresponding tensile force. The intensity of the parallel fluorescence polarization component decreases with increasing tension (adapted from van Mameren et al.91). (C) The four traps control four beads, and the XRCC4-XLF protein bridge formed by incubation is connected in the middle. The protein at the node is clearly excited (adapted from Brouwer et al.19). (D), 3 × 3 array trap intensity image shows the relationship between fluorescence intensity and tPSA concentration including beads coated with a single antigen and mixed antigen (adapted from Cao et al.20).

Close modal

The fluorescence emitted by the fluorophore is polarized under the excitation of the polarized laser. The polarized fluorescence method includes linear and circularly polarized excitation, which are used to study the orientation and rotation of molecules. The fluorescence polarization anisotropy method can measure the reorientation of a sample within an ultrashort time range between excitation and emission. Anisotropic information is obtained by detecting the ratio of the two polarized beams in the vertical and parallel directions. Mameren et al. recently combined polarization fluorescence with optical tweezers. The incident laser was excited to the DNA handle with dyes after being phase-delayed by the electro-optic modulator. A Wollaston prism was used to decompose the emitted light into two mutually polarized beams prior to entering the detector for intensity calculation and contrast. Under the tensile force of traps, the dsDNA helix gap was reduced, and the direction of the labeled points was changed accordingly. This device allowed the authors to determine that the average orientation of the inserted dye is perpendicular to the long axis of the DNA. After the DNA was overstretched, depolarization caused by the average tilt of the base pairs was observed (Fig. 10B). This work helped clarify the unique characteristics of DNA dynamics.91 

Besides single- and dual-trap, multi and array traps can also be combined with fluorescence. Brouwer et al. used a same laser to form four traps.19 Each of the two traps form a group, which is equivalent to two dual-trap systems arranged up and down. Each dual-trap binds with a DNA molecule. The two DNA fragments are entangled to repair under the action of XLF protein (Fig. 10C). This device validated the complex movement mechanism of XRCC4-XLF. The structures around the DNA molecule rejoin cleavage ends and quickly bind them together, thereby clarifying the involvement of NHEJ protein in the generation of chromosomal translocations. King et al. applied this same design to quantify the real-time tension of local molecular systems with 2 pN resolution. In their experiment, the short DNA molecule inserted with dye was used as a standard quantitative sensor, and the fluorescence intensity was determined by the tensile force it received. Such a fluorescence-force senor has rich applications; it can be used to measure the force in any system tethered by dsDNA, protein unfolding, chromosome mechanics, cell movement, and DNA molecular machinery, among others.92 

Array traps are modified on the incident laser by a spatial modulation holographic device to form an equally sized trap array. Cao et al. designed array traps measuring 3 × 3 size with nine beads fixed together and combined them with epi-fluorescence to achieve blood antigen detection.20 The sample solution was mixed with different size beads, each labeled with a specific antibody. The higher the concentration of antigen proteins in the blood, the stronger the intensity and the higher the imaging brightness. Simultaneous detection of multiple antigens could be achieved on the entire array (Fig. 10D). Liu et al. combined a holographic trap and confocal system to achieve optical control of zeta potential and local vibrational stimulation in target cells. The trap was responsible for injecting fluorescence sensors into cells through vibrational stimulation, and fluorescence is used for observation. The authors successfully injected the sensor within 30 min and revealed an effective injection rate of 80%.93 Lee et al. integrated a multi-color holographic trap, epi-fluorescence, and TIRF on a microscopic system equipped with multiple fluorescent laser sources and time-multiplexed. Each laser simultaneously acted as the trap and excitation source, capturing and displaying multi-color fluorescent bead arrays.21 This versatile trap and fluorescence system design integrated multiple optical devices but showed less than ideal measurement performance. The combination of multiple instruments is bound to damage various measurement advantages. Thus, building multi-functional and high-performance parallel-combined single-molecule system remains a challenging issue for researchers.

Many super-resolution fluorescence methods including STORM, PALM, RESOLFT, etc. are more advanced in imaging resolution and positioning accuracy. Despite these super resolution fluorescence methods are attractive and powerful, but not all of them are suitable for combining with optical tweezers, some unique set of limitations and blocked the combination with optical tweezers. Special fluorescence proteins are needed as marks to realize super-resolution, but the significant photobleaching is introduced by trap laser and system layout of optical tweezers limits the fluorescence structure. A suitable fluorescence method can be combined with optical tweezers is advanced in excitation laser structure but not the special label of sample. We still look forward to these excellent fluorescence technologies can be improved and combined with the optical tweezers, and promote the development of single-molecule.

The combination of optical tweezers and fluorescence single-molecule technology integrates two challenging measurement systems, namely, laser manipulation and fluorescence imaging, and enables the quantitative analysis and operation of the intrinsic structure and interactions of biomolecules. In this paper, the difference of combination method between the single-trap and dual-trap are analyzed from the fixed way of the test sample. Difficulties related to photobleaching, including the spatial separation method and the time separation method, are then analyzed. Finally, the methods and characteristics of various fluorescence microscopic technologies combined with trap system are analyzed by highlighting design ideas and technical difficulties. This work provides a source of understanding for researchers and a reference for workers who are about to engage in this field. The combination of optical tweezers and fluorescence technology can achieve the highest research results of current single-molecule measurement methods, thereby enabling researchers to achieve unprecedented precision and clarity in biological systems. The way to biological analysis, chemistry, medicine and toxicology has broadened. Commercialization of some mature devices has allowed researchers from a wide range of industries to experience this powerful tool. This technology may be expected to see further development as collaborative measurement capabilities improve and more novel fluorescent means are combined with optical tweezers. We hope more researchers can participate in this trend, introduce new ideas, continuously improve technology, and promote the development and innovation of life sciences.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This work was supported by the National Key Research and Development Program of China [grant numbers 2016YFB1102203, 2017YFF0107003].

1.
Greenleaf
WJ
,
Woodside
MT
,
Block
SM
.
High-resolution, single-molecule measurements of biomolecular motion
.
Annu. Rev. Biophys. Biomol. Struct.
2007
;
36
(
1
):
171
190
.
2.
Mehta
AD
,
Rief
M
,
Spudich
JA
 et al 
Single-molecule biomechanics with optical methods
.
Science
1999
;
283
(
5408
):
1689
1695
.
3.
Fang
J
,
Mehlich
A
,
Koga
N
 et al 
Forced protein unfolding leads to highly elastic and tough protein hydrogels
.
Nat. Commun.
2013
;
4
:
2974
.
4.
Lee
KS
,
Balci
H
,
Jia
H
 et al 
Direct imaging of single UvrD helicase dynamics on long single-stranded DNA
.
Nat. Commun.
2013
;
4
:
1878
.
5.
Ashkin
A
,
Dziedzic
JM
,
Yamane
T
.
Optical trapping and manipulation of single cells using infrared laser beams
.
Nature
1987
;
330
(
6150
):
769
771
.
6.
Ashkin
A
,
Dziedzic
JM
.
Optical trapping and manipulation of viruses and bacteria
.
Science
1987
;
235
(
4795
):
1517
1520
.
7.
Ashkin
A
.
History of optical trapping and manipulation of small-neutral particle, atoms, and molecules
.
IEEE J. Sel. Top. Quantum Electron.
2000
;
6
(
6
):
841
856
.
8.
Neuman
KC
,
Block
SM
.
Optical trapping
.
Rev. Sci. Instrum.
2004
;
75
(
9
):
2787
2809
.
9.
Whitley
KD
,
Comstock
MJ
,
Chemla
YR
.
Optical Tweezers: Methods and Protocols
.
NY
:
Springer New York
;
2017
. p.
183
256
.
10.
Weiss
S.
Fluorescence spectroscopy of single biomolecules
.
Science
1999
;
283
(
5408
):
1676
1683
.
11.
Joo
C
,
Balci
H
,
Ishitsuka
Y
 et al 
Advances in single-molecule fluorescence methods for molecular biology
.
Annu. Rev. Biochem.
2008
;
77
(
1
):
51
76
.
12.
Sun
LL
,
Su
YY
,
Gao
YJ
 et al 
Progresses of single molecular fluorescence resonance energy transfer in studying biomacromolecule dynamic process
.
Chin. J. Anal. Chem.
2018
;
46
(
6
):
803
813
.
13.
Lang
MJ
,
Fordyce
PM
,
Engh
AM
 et al 
Simultaneous, coincident optical trapping and single-molecule fluorescence
.
Nat. Methods
2004
;
1
(
2
):
133
139
.
14.
Eggeling
C
,
Widengren
J
,
Rigler
R
 et al 
Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis
.
Anal. Chem.
1998
;
70
(
13
):
2651
2659
.
15.
Heller
I
,
Sitters
G
,
Broekmans
OD
 et al 
Mobility analysis of super-resolved proteins on optically stretched DNA: Comparing imaging techniques and parameters
.
ChemPhysChem
2014
;
15
(
4
):
727
733
.
16.
van Dijk
MA
,
Kapitein
LC
,
van Mameren
J
 et al 
Combining optical trapping and single-molecule fluorescence spectroscopy: Enhanced photobleaching of fluorophores
.
J. Phys. Chem. B
2004
;
108
(
20
):
6479
6484
.
17.
Steven
CHU
.
Laser manipulation of atoms and particles
.
Science
1991
;
253
(
5022
):
861
866
.
18.
Shabestari
MH
,
Meijering
A
,
Roos
W
 et al 
Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods
.
Academic Press
;
2017
. p.
85
119
.
19.
Brouwer
I
,
Sitters
G
,
Candelli
A
 et al 
Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA
.
Nature
2016
;
535
(
7613
):
566
569
.
20.
Cao
D
,
Li
CY
,
Qi
CB
 et al 
Multiple optical trapping assisted bead-array based fluorescence assay of free and total prostate-specific antigen in serum
.
Sens. Actuators, B
2018
;
269
:
143
150
.
21.
Lee
SH
.
Optimal integration of wide field illumination and holographic optical tweezers for multimodal microscopy with ultimate flexibility and versatility
.
Opt. Express
2018
;
26
(
7
):
8049
8058
.
22.
Ali
MY
,
Homma
K
,
Iwane
AH
 et al 
Unconstrained steps of myosin vi appear longest among known molecular motors
.
Biophys. J.
2004
;
86
(
6
):
3804
3810
.
23.
Ferrer
JM
,
Lee
H
,
Chen
J
 et al 
Measuring molecular rupture forces between single actin filaments and actin-binding proteins
.
Proc. Natl. Acad. Sci. U. S. A.
2008
;
105
(
27
):
9221
9226
.
24.
Clancy
BE
,
Behnke-parks
WM
,
Andreasson
JOL
 et al 
A universal pathway for kinesin stepping
.
Nat. Struct. Mol. Biol.
2011
;
18
(
9
):
1020
1027
.
25.
Bianco
PR
,
Brewer
LR
,
Corzett
M
 et al 
Processive translocation and DNA unwinding by individual RecBCD enzyme molecules
.
Nature
2001
;
409
(
6818
):
374
378
.
26.
Handa
N
,
Bianco
PR
,
Baskin
RJ
 et al 
Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition
.
Mol. Cell
2005
;
17
(
5
):
745
750
.
27.
Bianco
PR
,
Bradfield
JJ
,
Castanza
LR
 et al 
Rad54 oligomers translocate and cross-bridge double-stranded DNA to stimulate synapsis
.
J. Mol. Biol.
2007
;
374
(
3
):
618
640
.
28.
Hilario
J
,
Amitani
I
,
Baskin
RJ
 et al 
Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules
.
Proc. Natl. Acad. Sci. U. S. A.
2009
;
106
(
2
):
361
368
.
29.
Pezza
RJ
,
Camerini-otero
RD
,
Bianco
PR
.
Hop2-Mnd1 condenses DNA to stimulate the synapsis phase of DNA strand exchange
.
Biophys. J.
2010
;
99
(
11
):
3763
3772
.
30.
Pang
Y
,
Song
H
,
Kim
JH
 et al 
Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution
.
Nat. Nanotechnol.
2014
;
9
(
8
):
624
630
.
31.
Ngo
TM
,
Zhang
Q
,
Zhou
R
 et al 
Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility
.
Cell
2015
;
160
(
6
):
1135
1144
.
32.
Grashoff
C
,
Hoffman
BD
,
Brenner
MD
 et al 
Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics
.
Nature
2010
;
466
(
7303
):
263
266
.
33.
Zhou
R
,
Kozlov
A
,
Roy
R
 et al 
SSB functions as a sliding platform that migrates on DNA via reptation
.
Cell
2011
;
146
(
2
):
222
232
.
34.
Funatsu
T
,
Harada
Y
,
Tokunaga
M
 et al 
Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution
.
Nature
1995
;
374
(
6522
):
555
559
.
35.
Beebe
DJ
,
Mensing
GA
,
Walker
GM
.
Physics and applications of microfluidics in biology
.
Annu. Rev. Biomed. Eng.
2002
;
4
(
1
):
261
286
.
36.
Squires
TM
,
Quake
SR
.
Microfluidics: Fluid physics at the nanoliter scale
.
Rev. Mod. Phys.
2005
;
77
(
3
):
977
1026
.
37.
Iwaki
M
,
Tanaka
H
,
Iwane
AH
 et al 
Cargo-binding makes a wild-type single-headed myosin-VI move processively
.
Biophys. J.
2006
;
90
(
10
):
3643
3652
.
38.
Fujita
K
,
Iwaki
M
,
Iwane
AH
 et al 
Switching of myosin-V motion between the lever-arm swing and Brownian search-and-catch
.
Nat. Commun.
2012
;
3
:
956
.
39.
Akiyoshi
B
,
Sarangapani
KK
,
Powers
AF
 et al 
Tension directly stabilizes reconstituted kinetochore-microtubule attachments
.
Nature
2010
;
468
(
7323
):
576
579
.
40.
Umbreit
NT
,
Miller
MP
,
Tien
JF
 et al 
Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation
.
Nat. Commun.
2014
;
5
:
4951
.
41.
Sarangapani
K
,
Duro
E
,
Deng
Y
 et al 
Sister kinetochores are mechanically fused during meiosis I in yeast
.
Science
2014
;
346
(
6206
):
248
251
.
42.
Bustamante
C
,
Chemla
YR
,
Moffitt
JR
.
High-resolution dual-trap optical tweezers with differential detection: Instrument design
.
Cold Spring Harb. Protoc.
2009
;
10
:http://cshprotocols.cshlp.org/content/2009/10/pdb.ip73.abstract.
43.
Abbondanzieri
EA
,
Greenleaf
WJ
,
Shaevitz
JW
 et al 
Direct observation of base-pair stepping by RNA polymerase
.
Nature
2005
;
438
(
7067
):
460
465
.
44.
Moffitt
JR
,
Chemla
YR
,
Izhaky
D
 et al 
Differential detection of dual traps improves the spatial resolution of optical tweezers
.
Proc. Natl. Acad. Sci. U. S. A.
2006
;
103
(
24
):
9006
9011
.
45.
Saito
K
,
Aoki
T
 et al 
Movement of single myosin filaments and myosin step size on an actin filament suspended in solution by a laser trap
.
Biophys. J.
1994
;
66
(
3
):
769
777
.
46.
Arai
Y
,
Yasuda
R
,
Akashi
KI
 et al 
Tying a molecular knot with optical tweezers
.
Nature
1999
;
399
(
6735
):
446
448
.
47.
Comstock
MJ
,
Whitley
KD
,
Jia
H
 et al 
Direct observation of structure-function relationship in a nucleic acid-processing enzyme
.
Science
2015
;
348
(
6232
):
352
354
.
48.
Duesterberg
VK
,
Fischer-hwang
IT
,
Perez
CF
 et al 
Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer
.
eLife
2015
;
4
.
49.
Ishijima
A
,
Kojima
H
,
Funatsu
T
 et al 
Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin
.
Cell
1998
;
92
(
2
):
161
171
.
50.
Komori
T
,
Nishikawa
S
,
Ariga
T
 et al 
Simultaneous measurement of nucleotide occupancy and mechanical displacement in myosin-V, a processive molecular motor
.
Biophys. J.
2009
;
96
(
1
):
L4
L6
.
51.
Heller
I
,
Sitters
G
,
Broekmans
OD
 et al 
STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA
.
Nat. Methods
2013
;
10
(
9
):
910
916
.
52.
Moffitt
JR
,
Chemla
YR
,
Smith
SB
 et al 
Recent advances in optical tweezers
.
Annu. Rev. Biochem.
2008
;
77
(
1
):
205
228
.
53.
Murade
CU
,
Subramaniam
V
,
Otto
C
 et al 
Interaction of oxazole yellow dyes with DNA studied with hybrid optical tweezers and fluorescence microscopy
.
Biophys. J.
2009
;
97
(
3
):
835
843
.
54.
Smith
SB
,
Cui
Y
,
Bustamante
C
.
Overstretching B-DNA: The elastic response of individual double-stranded and single-stranded DNA molecules
.
Science
1996
;
271
(
5250
):
795
799
.
55.
King
GA
,
Peterman
EJG
,
Wuite
GJL
.
Unravelling the structural plasticity of stretched DNA under torsional constraint
.
Nat. Commun.
2016
;
7
.
56.
Biebricher
AS
,
Heller
I
,
Roijmans
RFH
 et al 
The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics
.
Nat. Commun.
2015
;
6
:
7304
.
57.
van Mameren
J
,
Modesti
M
,
Kanaar
R
 et al 
Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers
.
Biophys. J.
2006
;
91
(
8
):
L78
L80
.
58.
Candelli
A
,
Holthausen
JT
,
Depken
M
 et al 
Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution
.
Proc. Natl. Acad. Sci. U. S. A.
2014
;
111
(
42
):
15090
15095
.
59.
Gao
Y
,
Zorman
S
,
Gundersen
G
 et al 
Single reconstituted neuronal SNARE complexes zipper in three distinct stages
.
Science
2012
;
337
(
6100
):
1340
1343
.
60.
Brouwer
I
,
Giniatullina
A
,
Laurens
N
 et al 
Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes
.
Nat. Commun.
2015
;
6
:
8387
.
61.
Min
TL
,
Mears
PJ
,
Chubiz
LM
 et al 
High-resolution, long-term characterization of bacterial motility using optical tweezers
.
Nat. Methods
2009
;
6
(
11
):
831
835
.
62.
Mears
PJ
,
Koirala
S
,
Rao
CV
 et al 
Escherichia coli swimming is robust against variations in flagellar number
.
eLife
2014
;
3
.
63.
Gross
P
,
Farge
G
,
Peterman
EJ
 et al 
Single Molecule Tools, Part B: Super-resolution, Particle Tracking, Multiparameter, and Force Based Methods
.
Academic Press
;
2010
. p.
427
453
.
64.
Biebricher
A
,
Wende
W
,
Escudé
C
 et al 
Tracking of single quantum dot labeled EcoRV sliding along DNA manipulated by double optical tweezers
.
Biophys. J.
2009
;
96
(
8
):
L50
L52
.
65.
Harada
Y
,
Funatsu
T
,
Murakami
K
 et al 
Single-molecule imaging of RNA polymerase-DNA interactions in real time
.
Biophys. J.
1999
;
76
(
2
):
709
715
.
66.
Hohng
S
,
Zhou
R
,
Nahas
MK
 et al 
Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday junction
.
Science
2007
;
318
(
5848
):
279
283
.
67.
Schakenraad
K
,
Biebricher
AS
,
Sebregts
M
 et al 
Hyperstretching DNA
.
Nat. Commun.
2017
;
8
(
1
):
2197
.
68.
Comstock
MJ
,
Ha
T
,
Chemla
YR
.
Ultrahigh-resolution optical trap with single-fluorophore sensitivity
.
Nat. Methods
2011
;
8
(
4
):
335
340
.
69.
Brau
RR
,
Tarsa
PB
,
Ferrer
JM
 et al 
Interlaced optical force-fluorescence measurements for single molecule biophysics
.
Biophys. J.
2006
;
91
(
3
):
1069
1077
.
70.
Visscher
K
,
Gross
SP
,
Block
SM
.
Construction of multiple-beam optical traps with nanometer-resolution position sensing
.
IEEE J. Sel. Top. Quantum Electron.
1996
;
2
(
4
):
1066
1076
.
71.
Sirinakis
G
,
Ren
Y
,
Gao
Y
 et al 
Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy
.
Rev. Sci. Instrum.
2012
;
83
(
9
).
72.
Suksombat
S
,
Khafizov
R
,
Kozlov
AG
 et al 
Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways
.
eLife
2015
;
4
.
73.
Galletto
R
,
Amitani
I
,
Baskin
RJ
 et al 
Direct observation of individual RecA filaments assembling on single DNA molecules
.
Nature
2006
;
443
(
7113
):
875
878
.
74.
Dong
J
,
Castro
CE
,
Boyce
MC
 et al 
Optical trapping with high forces reveals unexpected behaviors of prion fibrils
.
Nat. Struct. Mol. Biol.
2010
;
17
(
12
):
1422
1430
.
75.
van Mameren
J
,
Modesti
M
,
Kanaar
R
 et al 
Counting RAD51 proteins disassembling from nucleoprotein filaments under tension
.
Nature
2008
;
457
(
7230
):
745
748
.
76.
van den Broek
B
,
Noom
MC
,
van Mameren
J
 et al 
Visualizing the formation and collapse of DNA toroids
.
Biophys. J.
2010
;
98
(
9
):
1902
1910
.
77.
Noom
MC
,
van den Broek
B
,
van Mameren
J
 et al 
Visualizing single DNA-bound proteins using DNA as a scanning probe
.
Nat. Methods
2007
;
4
(
12
):
1031
1036
.
78.
King
GA
,
Gross
P
,
Bockelmann
U
 et al 
Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy
.
Proc. Natl. Acad. Sci. U. S. A.
2013
;
110
(
10
):
3859
3864
.
79.
Cardinale
M
.
Scanning a microhabitat: Plant-microbe interactions revealed by confocal laser microscopy
.
Front. Microbiol.
2014
;
5
:
94
.
80.
Leijnse
N
,
Oddershede
LB
,
Bendix
PM
.
Helical buckling of actin inside filopodia generates traction
.
Proc. Natl. Acad. Sci. U. S. A.
2015
;
112
(
1
):
136
141
.
81.
Bornschlogl
T
,
Romero
S
,
Vestergaard
CL
 et al 
Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip
.
Proc. Natl. Acad. Sci. U. S. A.
2013
;
110
(
47
):
18928
18933
.
82.
Whitley
KD
,
Comstock
MJ
,
Chemla
YR
.
Ultrashort nucleic acid duplexes exhibit long wormlike chain behavior with force-dependent edge effects
.
Phys. Rev. Lett.
2018
;
120
(
6
):
68102
.
83.
Kurniawan
N
,
Vos
B
,
Biebricher
A
 et al 
Fibrin networks support recurring mechanical loads by adapting their structure across multiple scales
.
Biophys. J.
2016
;
111
(
5
):
1026
1034
.
84.
Ramesh
P
,
Baroji
YF
,
Reihani
SNS
 et al 
FBAR syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner
.
Sci. Rep.
2013
;
3
:
1565
.
85.
Roy
D
,
Mondal
D
,
Goswami
D
.
Elucidating microscopic structure and dynamics in optically tweezed environments
.
Chem. Phys. Lett.
2015
;
621
:
203
208
.
86.
Sparkes
I
,
White
RR
,
Coles
B
 et al 
The Plant Endoplasmic Reticulum: Methods and Protocols
.
NY
:
Springer New York
;
2018
. p.
167
178
. http://link.springer.com/10.1007/978-1-4939-7389-7.
87.
Lee
JY
,
Wang
F
,
Fazio
T
 et al 
Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains
.
Biochem. Biophys. Res. Commun.
2012
;
426
(
4
):
565
570
.
88.
Simmert
S
,
Abdosamadi
MK
,
Hermsdorf
G
 et al 
LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional microtubule imaging
.
Opt. Express
2018
;
26
(
11
):
14499
14513
.
89.
Deng
Y
,
Asbury
CL
.
Optical Tweezers: Methods and Protocols
.
NY
:
Springer New York
;
2017
. p.
437
467
.
90.
Lin
CT
,
Tritschler
F
,
Lee
KS
 et al 
Single-molecule imaging reveals the translocation and DNA looping dynamics of hepatitis C virus NS3 helicase
.
Protein Sci.
2017
;
26
(
7
):
1391
1403
.
91.
van Mameren
J
,
Vermeulen
K
,
Wuite
GJL
 et al 
A polarized view on DNA under tension
.
J. Chem. Phys.
2018
;
148
(
12
).
92.
King
G
,
Biebricher
AS
,
Heller
I
 et al 
Quantifying local molecular tension using intercalated DNA fluorescence
.
Nano Lett.
2018
;
18
(
4
):
2274
2281
.
93.
Liu
H
,
Maruyama
H
,
Masuda
T
 et al 
Vibration-assisted optical injection of a single fluorescent sensor into a target cell
.
Sens. Actuators, B
2015
;
220
:
40
49
.

Guoteng Ma, School of Precision Instrument and Opto- Electronics Engineering, Tianjin University, State Key Laboratory of Precision Measuring Technology and Instruments. Mr. Ma is studying for PhD. His research interests include: optical tweezers for single-molecule, fluorescence microscopy and 2D materials.

Chunguang Hu, Associate Professor, School of Precision Instrument and Opto-Electronics Engineering, Tianjin University, State Key Laboratory of Precision Measuring Technology and Instruments. Prof. Hu received his PhD degree in 2007 and is currently working at Tianjin University. His research interests include: optical characterization of nano/micro-structures on surfaces, nano/micro scale sensors and single molecule technology for biology.