Fig. 1. Basic principle of optical tweezers. (a) Lateral gradient force on particles larger than wavelength in non-uniform field^[20]. (b) Axial gradient force towards trapping laser focus^[20]. (c) Ray model for optical force^[21]. (d) Dipoles smaller than wavelength are subject to scattering forces (Fscat, red arrows) and gradient forces (Fgrad, black arrows)^[12]

Fig. 2. Optical trapping strategies for single-molecule studies. (a) Single beam optical trap. A single DNA molecule is tethered between trapped bead and bottom glass slide^[40]. (b) Trap-pipette geometry. A molecule is attached to a second bead held by suction into a micropipette^[40]. (c) Dual-trap “dumbbell” assay. A molecule is tethered between two trapped beads^[40]. (d) Experimental configuration to measure supercoil torque on a DNA molecule with AOT^[42]. (e) Quadruple-trap optical tweezers manipulate dual DNA molecules^[43]

Fig. 3. Schematics of dual-trap optical tweezers for single-molecule study. (a) Layout of dual-trap optical tweezers. (b) Schematics of single-molecule essay. Preassembled SNARE complex was crosslinked to 2260 bp DNA handle through disulfide bond. (c) Extension-time trajectories of single SNARE complexes under constant trap separation. Ideal state transitions derived from hidden-Markov model (HMM) are expressed in red lines. Configurations for each state are represented at the bottom

Fig. 4. Power spectral density curves when a 1.9-µm-diameter bead is trapped (red), when laser is on without bead (blue), and when there is no laser beam (rose red)

Fig. 5. Measurement of dsDNA by dual-trap optical tweezers. (a) Schematic diagram. (b) Resolution measurements under the sampling frequency of 100 kHz. Pairwise distance distribution of (b) suggests a step size of 4.6 Å (1 Å=0.1 nm) (c) and 2.3 Å (d)^[53]

Fig. 6. Central dogma in molecular biology. (a) Overview of central dogma. DNA is self-replicating and information contained in DNA sequence is transcribed into RNA. RNA message sequence is translated into sequence of polypeptide by ribosome. Polypeptides are folded into proteins. (b) Main parameters of double-stranded DNA and protein secondary structure

Fig. 7. Study of protein folding. (a) Schematic diagram of single-molecule research on monomer and dimer PrP misfolding^[73]. (b) With force held constant at 9.1 pN, protein jumps between unfolded state (U) and natively folded state (N)^[74]. (c) Unfolding (black) and refolding (red) FECs of PrP dimers show formation of stable nonnative structures^[73]. (d) Transition path time (t_tp) for misfolding measured from constant-force trajectories for I_D1↔U^[73]. (e) FECs of T4 lysozyme unfolding (red) and refolding (blue) on ribosome^[80]. (f) Schematic diagram of single-molecule study of advanced structure of EF-G^[81]. (g) Representative force extension curves from pulls of EF-G^[81]. (h) Ribosome and TF reduce inter-domain misfolding: top picture is G-domain refolding against constant force of 3.5 pN, and bottom picture is refolding in presence of TF and ribosome (452RNC)^[82]

Fig. 8. Study on DNA-protein interaction. (a) Schematics of RNAP dumbbell assay^[88]. (b) Step records for single molecular RNAP transcribing under 18 pN^[88]. (c) Average autocorrelation function derived from position histograms of RNAP transcription exhibits periodicity at multiples of step size (inset). Its power spectrum shows a peak at dominant spatial frequency, corresponding to (0.37±0.06) nm^[88]. (d) Biotinylated RNAP is bridged to biotinylated 1.5 kb DNA^[89]. (e) Representative traces obtained under mechanical force (left). Mean residence time histogram (right) is calculated by averaging the time spent at each position in the repeat across all traces at all conditions. Pause sites are marked, and pause-free regions are shaded^[89]

Fig. 9. Mechanochemical properties of molecular motors. (a) Schematic of dual-trap optical tweezers for DNA fork unwinding. wtRep structure in “closed” conformation shows its four subdomains, and structural model of Rep Δ2B with 2B is replaced by three glycine residues^[103]. (b) Trace of processive DNA fork unwinding by Rep Δ2B monomer. Zoom-in plot shows two rounds of activity: protein unwinding (U) ends either by mid-fork dissociation (D) or dissociation at fork base after three rounds of unwinding (U) and rezipping (Z)^[103]. (c) Experimental geometry of high-resolution packaging assay^[105]. (d) Sample traces displaying individual packaging cycles at various levels of capsid filling. Stepwise fit highlights dwells and bursts in red and green, respectively^[105]. (e),(f) Characteristics of packaging at low capsid filling (e) and high capsid filling (f) in bacteriophage packaging^[105]

Fig. 10. CpxI stabilizes partially folded SNARE complex^[111]. (a) Extension-time trajectories of single SNARE complex under constant trap separation in presence of CpxI. Arrows indicate administration of full-length CpxI molecules. (b) Enlarged views of region marked by dashed blue rectangles in (a). SNARE configurations correspond to each state. (c) Probability distributions of extensions correspond to traces in (a) in presence (red circles) and absence (black triangles) of CpxI and their best fits by a sum of four Gaussian functions (red and black lines). This suggests that in presence of full-length CpxI, SNARE complex is locked into C-terminal assembly-inhibited state

Fig. 11. Laser Raman spectroscopy tweezers. (a) Schematic of dual-trap Raman tweezers^[132]. (b) Schematic of Raman spectroscopy system with dual-spot optical trap^[134]. (c) Time-lapse Raman spectra of daughter cell and parent of budding yeast cell in dual traps^[132]. (d) Raman spectra of equilibrium (gray line) and stretched (dark line) states of RBC^[134]

Fig. 12. Combination of optical tweezers and single-molecule fluorescence detection. (a) Single trap with total internal reflection fluorescence microscopy with which one UvrD monomer translocates along ssDNA^[139]. (b) Dual-trap and epifluorescence configuration, showing labeled RAD51 bound to DNA and fluorescence collected from dye (green circles)^[143]. (c) Schematic of fluorescently labeled SSB, SSB_f, ssDNA wrapping experiment^[144]. (d) UvrD unwounds dsDNA events. White dashed line indicates projected location of junction^[139]. (e) Kymograph and fluorescence intensity trace of RAD51-dsDNA complex, held at fixed length^[140]. (f) Representative traces of fluorescence and DNA extension measured during SSB wrapping ssDNA^[144]

Fig. 13. Combination of ultra-high resolution imaging technology with tweezers^[147]. (a) Beam path in experimental setup of STED combined confocal microscope and dual-trap optical tweezers. (b) Confocal microscopy image of fluorescent labeled λDNA and protein. (c) Two optically trapped microspheres tethered by DNA molecule, where excitation (EXC) beam (and superimposed STED beam) that is scanned over DNA is shown. (d) STED nanoscopy of SYTOX Red on optically stretched DNA

Fig. 14. Nano-plasmonic tweezers. (a) Schematic of trapping metallic particles by SPP virtual probe^[151]. (b) Distribution of electric field intensity and Poynting vector in horizontal plane of focused plasmonic tweezers^[151]. (c) Gold particles trapped by focused plasmonic tweezers^[151]. (d) Schematic of double-nanohole optical tweezers^[161]. (e) SEM image of double-nanohole^[161]. (f) Time track of BSA molecular optical power in different states transmitted through double-nanohole^[161]. (g) Comparison of cumulative probability of unzipping time ∆t for p53-DNA complex and DNA alone^[162]