High-Resolution "Fleezers": Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection

Abstract

Recent advances in optical tweezers have greatly expanded their measurement capabilities. A new generation of hybrid instrument that combines nanomechanical manipulation with fluorescence detection-fluorescence optical tweezers, or "fleezers"-is providing a powerful approach to study complex macromolecular dynamics. Here, we describe a combined high-resolution optical trap/confocal fluorescence microscope that can simultaneously detect sub-nanometer displacements, sub-piconewton forces, and single-molecule fluorescence signals. The primary technical challenge to these hybrid instruments is how to combine both measurement modalities without sacrificing the sensitivity of either one. We present general design principles to overcome this challenge and provide detailed, step-by-step instructions to implement them in the construction and alignment of the instrument. Lastly, we present a set of protocols to perform a simple, proof-of-principle experiment that highlights the instrument capabilities.

Keywords: Confocal microscopy; FRET; Fleezers; Förster resonance energy transfer; Optical trapping; Optical tweezers; Single-molecule fluorescence.

Fig. 1

Examples of experiments with combined high-resolution optical trap and fluorescence (not to scale). Left panels: Polystyrene microspheres (grey) are held in optical traps (orange cones), tethered by an engineered DNA molecule (blue) containing a variable central segment flanked by long double-stranded DNA (dsDNA) handles. Fluorophores are excited by a green laser (green cone). Right panels: Time traces showing simultaneous measurement of fluorescence and tether extension. (a) Oligonucleotide hybridization. Short oligonucleotides (blue line) labeled with a fluorophore (green disk) bind and unbind to a complementary ssDNA section in the center of the tethered DNA. The fluorescence and change in tether extension upon hybridization are recorded simultaneously. (b) Single-stranded DNA binding protein wrapping dynamics. A tethered DNA molecule containing a short ssDNA region is labeled with a FRET acceptor at the ss-dsDNA junction (red disk). An E. coli single-stranded DNA binding protein (SSB, cyan) labeled with a FRET donor (green disk) binds to and wraps ssDNA around itself. Simultaneous measurement of FRET efficiency and tether extension enables determination of both the position of SSB along the tether and the amount of ssDNA wrapped. The SSB can transiently wrap and unwrap ssDNA under tension (e.g., t = 20 s), and can diffuse one-dimensionally along the ssDNA by reptation (t = 70 s) (data reproduced from ref. [22] with permission from eLife Sciences Publications). (c) UvrD helicase conformational and unwinding dynamics. A tethered DNA molecule contains a hairpin and short ssDNA protein loading site. E. coli UvrD helicase (cyan, blue, grey, and green) is labeled with FRET donor and acceptor pair to differentiate between two possible conformational states: “Open” and “Closed.” Simultaneous measurement of FRET efficiency and number of DNA base pairs of the hairpin unwound by the helicase enables correlation of the conformation with the activity of the helicase. Changes in UvrD conformational state correspond to switches between unwinding and rezipping of the DNA hairpin (reproduced from ref. [23] with permission from AAAS). The proteins in this figure were prepared with VMD [48] using PDB entries 1EYG, 2IS2, and 3LFU

Fig. 2

Protocol summary. (a) Sequence of major steps involved in assembling and aligning the instrument. (b) Materials for the trap + fluorescence assay are prepared in parallel, including two sets of functionalized beads: anti-digoxigenin (ADig) beads and streptavidin (Strep) beads, and stock solutions of glucose oxidase + catalase (GOx) and trolox (TX). (c) Major steps involved in setting up a trap + fluorescence assay

Fig. 3

Detailed layout of the instrument (to scale). FC fiber clamp; ISO optical isolator; HW half-wave plate; PBS polarizing beam-splitting cube; BD beam dump; AOM acousto-optic modulator; L lens; M mirror; BS beam-splitter; QPD quadrant photodiode; DM dichroic mirror; FO front objective; BO back objective; F filter; RL relay lens; TL tube lens; ND neutral density filter; PD photodiode; SM steerable mirror; PSD position-sensitive detector; PH pinhole; APD avalanche photodiode. Planes conjugate to AOM1 are indicated by an asterisk (*), those conjugate to SM are indicated by a double cross (‡), and those conjugate to the sample plane are indicated by an x (×). Double-sided arrows at L5 and L8 indicate adjustable translational stages. The circular arrow at SM indicates a steerable mirror. Dotted lines indicate the front and back focal planes of FO and BO

Fig. 4

Photograph of instrument. The instrument is organized into three separate “modules” (Trap, Fluorescence, and Bright-field), indicated by the colored dotted lines. Major components of the instrument (AOMs, objectives, bead position detectors, and APDs) are labeled

Fig. 5

Interlacing and timesharing of optical trap and fluorescence excitation lasers, and synchronization of lasers with data acquisition timing. Two optical traps (orange) are created in sequence during two thirds of the interlacing period by time-sharing. The trap AOM (AOM1) switches between two deflection angles (traps in each interval are set to different intensities for clarity in the figure). Trap data acquisition occurs at time points centered on each trap interval. “×” and “+” denote the time points for the first and second trap, respectively. The rising edge of a digital pulse (black) is synchronous with the trap data acquisition timing (red vertical arrows). The fluorescence excitation (green) is only ON during the last third of the interlacing period while the trap is OFF. There are 625-ns delays (grey shaded regions) between turning OFF (ON) the optical traps and turning ON (OFF) the fluorescence excitation. A digital pulse (magenta) synchronous with the APD data acquisition timing is centered on the excitation laser interval. Fluorescence emission signals are only collected during this third time interval (red horizontal arrow). Laser intensities in the plot are measured by feedback photodetectors QPD1 and PD (see Fig. 3), and digital pulses synchronous with data acquisition timing are output directly from the DAQ card trap input timing debug (TDB) and APD gate timing debug (ADB) lines (see Fig. 10). All are recorded using a digital oscilloscope

Fig. 6

Effect of intensity feedback on trap performance. (a) The trapping beam power changes significantly as the trapping AOM deflects the beam over a range of distances in the specimen plane (blue), but remains constant with feedback ON (red). (b) Feedback stabilizes the trap laser intensity against drift over long time periods (red, feedback ON; blue, feedback OFF). (c) Noise power spectrum of laser intensity. Use of feedback reduces low-frequency noise from the trapping laser by up to six orders of magnitude (red, feedback ON; blue, feedback OFF) (reproduced from ref. [16] with permission from Nature Publishing Group)

Fig. 7

Comparison of IR-enhanced QPD to PSD for IR laser detection. The trapping laser power measured by an IR-enhanced QPD (blue) and PSD (red) during its ON/OFF interlacing cycle is shown. The PSD exhibits parasitic low-pass filtering, characterized by long rise and fall times (reproduced from ref. [16] with permission from Nature Publishing Group)

Fig. 8

Noise power spectra from commercial and custom-built RF synthesizers. The custom-built RF synthesizer (blue) exhibits lower noise than the commercial synthesizer (red) by up to four orders of magnitude (reproduced from ref. [16] with permission from Nature Publishing Group)

Fig. 9

Transmission efficiency of trapping AOM as a function of input RF power. The intensity of the first-order diffracted beam relative to the total intensity input to the AOM increases with RF power. High input RF power can damage the AOM (red hatched region)

Fig. 10

Input/output architecture of the FPGA-based DAQ card. Thicker lines refer to groups of wires. The FPGA uses 20 DO lines to communicate with the trap RF synthesizer, including six for individual bits denoting the address byte (AD) and eight for individual bits denoting the data byte (DA). The output RF signal from the synthesizer goes to an amplifier and then to the trap AOM. Three separate debugging DO lines (TDB, EDB, and ADB) are used for synchronizing detection input timing with the interlacing cycle (see Subheadings 3.2, 3.3, and 3.4). One of the FPGA-controlled AO lines is used to output an analog fluorescence signal (AFL) for aligning the instrument (see Subheading 3.4). The FPGA controls an additional set of AIO lines in an expansion chassis. This chassis has one AI line used for the fluorescence feedback PD, and one AO line that is used, along with a DO line from the FPGA, to control the fluorescence RF synthesizer

Fig. 11

Assembled RF synthesizer board. A ribbon cable (bottom, digital lines labeled) and a coaxial cable (FSK, top) carry digital signals to the board directly from the FPGA (see Fig. 10). The temperature-compensated crystal oscillator (TCXO) is mounted to a 14-pin DIP socket on the board. A coaxial cable carries the filtered RF output signal from the board to the amplifier (top left, labeled IOUT1 on board; the second cable is ignored). Power is supplied to the board from a single 3.3 VDC source (bottom left)

Fig. 12

Scheme for writing data to RF synthesizer board. Multiple bytes of data are sequentially transferred to a buffer by setting data and address DO lines and then sending a TTL pulse to the write (WR) line. After all necessary changes are made, they are all activated simultaneously by sending a pulse to the update clock (UDC) signal line, which initiates a simultaneous transfer of the buffer memory to the active memory

Fig. 13

Assembly of laminar flow chamber. (a) Expanded view of the “Parafilm sandwich” that comprises the chamber. A piece of Parafilm with flow channels cut into it is placed on a coverslip with eight holes cut into it. Two glass capillaries span the Parafilm to connect the bottom and top channels to the large central channel. A coverslip with no holes is then placed on top of the Parafilm to form the assembled chamber. (b) A fully assembled flow chamber. (c) A flow chamber mounted on an anodized aluminum bracket, held in place by two acrylic mounts. Four holes on either side of the mount are aligned with the holes of the coverslip. A short length of Tygon tubing is threaded through a set screw, and a longer stretch of polyethylene (PE) tubing is inserted into the Tygon tubing. Eight threaded set screws are prepared and screwed into the eight holes in the aluminum bracket to serve as inlet and outlet channels for the flow chamber. (d) Photograph of an assembled and mounted flow chamber

Fig. 14

Synchronization of frequency and amplitude switching of the RF signal. (a) RF signals for the two traps are shown with both frequency and amplitude different by a factor of 2 for clarity (trap 1, 45 MHz; trap 2, 90 MHz). With an appropriately programmed delay between amplitude and frequency signals from the FPGA, the switch happens synchronously (top panel). When there is no programmed delay, the frequency can switch before (middle panel) or after (bottom panel) the amplitude. (b) Traps 1 and 2 during the interlacing cycle, where the RF amplitude is chosen such that both traps have the same intensity. The dotted line indicates when the transition between trap 1 and trap 2 occurs. With the programmed delay between amplitude and frequency switching, the change from trap 1 to trap 2 occurs without any change in intensity (top panel). With no programmed delay, the intensity of trap 2 either drops (middle panel) or rises (bottom panel) to effectively “kick” the bead held in this trap

Fig. 15

Diffraction pattern produced by an AOM (either trap or fluorescence AOM). (a) When the AOM is OFF, only the zeroth order (undiffracted) beam is observed. (b) When the AOM is ON, several orders of diffracted beams are observed. The +1 order beam is the one used for trapping

Fig. 16

Images of trapped polystyrene beads using fluorescence imaging and detection laser scan. (a) Deflection in the x direction of the fluorescence excitation laser as it is scanned across the two trapped beads. (b) Deflection in the y direction of the excitation laser as it is scanned across the beads (blue denotes negative values; red, positive values). (c) Image of fluorescence intensity of two beads in Trap 1 and 2 recorded by the APDs. The signals result from autofluorescence of the beads (adsorbates can also give a signal)

Fig. 17

Adjustment of confocal spot focal depth. (a) Relative fluorescence intensity from a trapped fluorescent bead as the focal depth of the confocal spot is scanned (by scanning lens L8 along beam path). The fluorescence intensity drops significantly as the spot is moved away from the plane of the trapped beads. (b) PSD voltage-to-bead position conversion factors, α, derived from calibration using the excitation laser as a detection laser as the focal depth of the confocal spot is scanned. The conversion factors α are minimized when the confocal spot lies in the same plane as the trapped beads. Note: (a) and (b) were not performed for the same instrument alignment and disagree slightly

Fig. 18

Adjustment of front objective (FO) correction collar (collar shown in Fig. 20a). Relative fluorescence intensity and trap stiffnesses in the x and y directions from a trapped fluorescent bead as the correction collar is adjusted. The fluorescence intensity and trap stiffness do not reach a maximum at the same collar position likely due to chromatic aberrations in the objectives

Fig. 19

Construction of DNA substrate. Schematic depicting the major steps involved in preparing the DNA construct. The handles are first prepared by PCR of template DNA (pBR322 and λ DNA) using primers (FWD and REV) with either a biotin moiety (BIO, Left Handle) or a digoxigenin moiety (DIG, Right Handle) attached to the 5′ ends of the FWD primers. The two handles are digested by restriction enzymes (PspGI and TspRI) to produce 5′ and 3′ overhangs. The digested Left Handle is then ligated using T4 ligase to the short Insert containing a phosphate group on its 5′ end. Finally, the Right Handle is ligated to this product (Left Handle + Insert) to produce the final construct

Fig. 20

Alignment of flow chamber to front and back objectives. (a) Photograph of a mounted flow chamber resting on the motorized sample stage via a lens post, with the chamber between the front and back objectives. (b) and (c): Trapping laser beam profiles on the CCD camera when the chamber is misaligned in the y direction (b) and properly aligned (c). (d) and (e): Power spectra of trapped bead motion in y when the chamber is misaligned in the y direction (d) and properly aligned (e)

Fig. 21

Laminar flow cell layout sequence of steps in trap + fluorescence measurement. The flow chamber consists of top (yellow) and bottom (green) channels in which anti-digoxigenin (ADig) and DNA-coated streptavidin (DNA) beads flow, and a central channel comprised of two parallel laminar flow streams containing “blank” buffer (red) and the sample (blue). (Food dye was used in the photograph to show the different channels.) Within the flow chamber, ADig beads flow through the top channel (yellow) and out of the top capillary (black vertical lines) into the sample stream (blue), where it is captured by trap 1 (position 1). The chamber stage is then translated such that the trapped bead remains in the blank buffer stream (red) and is positioned near the bottom capillary (position 2). Here, DNA-coated streptavidin beads (DNA beads) are flowed through the bottom channel (green) and out of the bottom capillary, where one of them is captured by trap 2. When both beads are trapped, the stage is translated until the trapped beads are upstream of the capillaries (position 3). Here the traps are first calibrated, then an offset curve is taken (see Subheading 3.11). The trapped beads are then moved close to one another to form a single DNA tether. A F-X curve is taken to verify proper elastic behavior of the tethered molecule. Finally, the stage is translated such that the trapped beads are moved into the sample stream, which contains 1 nM fluorescently labeled oligonucleotide (position 4). At this position the excitation laser (green cone) is turned ON, and the binding and unbinding of oligonucleotides is observed both by the fluorescence signal and the change in extension of the traps. Whenever possible during this sequence, solution is flowed in the two central streams in order to maintain laminar flow (photo reproduced from ref. [14] with permission from eLife Sciences Publications)

Fig. 22

Obtaining a force-extension (F-X) curve of a DNA tether. (a) Recorded voltage from the bead position QPD as one trapped bead (in trap 1) is moved relative to the other trapped bead (in trap 2) (i.e., a blank F-X curve). The voltage offset V_off in the QPD output signal for trap 1 (red) and 2 (blue) depends on the separation between the two traps. Offset values obtained at the fixed positions where the traps are calibrated are also shown (× and +). (b) F-X curves of the DNA construct (Fig. 19) with either the “fixed” offset value (black and magenta) or the variable (separation-dependent) offset removed (red and blue). The data recorded from each trap are plotted separately for clarity. Only the F-X curves with a separation-dependent offset removed display the correct behavior. (c) The final F-X curve is plotted with the extensible wormlike chain (XWLC) model [49, 50]. The final F-X curve (cyan) is obtained by averaging the force from traps 1 and 2, where each has its variable offset removed individually. This averaged F-X curve is overlaid on the XWLC model (black dotted line) by shifting the curve along the extension axis by a small, fixed value resulting from uncertainty in the diameters of the trapped beads. The parameters used for the XWLC model for dsDNA and ssDNA are: persistence lengths P_ds = 50 nm, P_ss = 1 nm, helix rises h_ds = 0.34 nm/bp, h_ss = 0.59 nm/bp, and stretch moduli S_ds = S_ss = 1100 pN [–53]