Ultrahigh-resolution optical trap with single-fluorophore sensitivity

Abstract

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore-labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).

Figure 1

Combined ultra-high resolution optical trap and single-molecule fluorescence microscope setup. (a) Schematic of experimental setup showing dual optical traps (orange cones) trapping two beads with DNA tethered between them and confocal laser excitation and detection (green cone) measuring fluorescence from a single fluorophore-labeled molecule (magenta disk) bound to the DNA. Bead-DNA attachments are made via biotin (DNA) - streptavidin (bead) and digoxigenin (DNA) - anti-digoxigenin (bead) linkages. (b) Instrument layout showing optical paths for 1064-nm trapping laser (orange), 532-nm fluorescence excitation laser (green), collected fluorescence (magenta) and blue light-emitting diode (LED) for brightfield imaging (blue). Lasers are interlaced by acousto-optic modulators (AOM1 and 2 interlaced the trap and fluorescence lasers respectively) driven by radio frequency (RF) synthesizers directly controlled by a field programmable gate array (FPGA) chip-based data acquisition and control pc card. Synchronous with laser modulation, the FPGA reads three quadrant photodiodes (QPD) that measure trapped bead positions (QPD1) and trap and fluorescence excitation laser intensities (QPD2 and QPD3 respectively, enabling laser intensity stabilization) along with a single photon counting avalanche photodiode (APD) measuring fluorescence. Additional component abbreviations: dichroic mirror (D), filter (F), objective lens (O), pin hole filter (P), piezo mirror stage (PM), sample chamber (S), telescope (T). Conjugate image planes indicated by *. See Supplementary Note online for a complete parts list.

Figure 2

Interlacing and timesharing of optical trap and fluorescence excitation lasers. Two optical traps are created in sequence during time intervals A and B via the trap AOM switching between two trap laser intensities and deflection angles (traps in intervals A and B were set to different intensities for clarity in the figure). Trap data acquisition occurs at time points indicated by ‘x’ and ‘+’ for traps 1 and 2 respectively. The fluorescence excitation laser is only ON during time interval C while the trap laser is OFF. There are 625 ns delays (hatched intervals) between switching optical traps OFF (ON) and fluorescence excitation ON (OFF). Fluorescence is only collected during time interval C. Laser intensities in the plots were measured by photodetectors and recorded by a digital oscilloscope.

Figure 3

Single fluorophore-labeled oligonucleotides hybridization experiment. (a) Schematic of experimental setup. Two beads held in dual traps are tethered together by dsDNA (3 kbp) with a short ssDNA (19 nt) portion near the center. ssDNA probe strands diffuse in the surrounding solution and bind-unbind to the complementary tethered ssDNA. (b) Fluorescence image of the experiment acquired by the instrument. A probe strand bound to the tethered DNA is observed as a fluorescent spot localized between the two beads. (c) Fluorescence vs. time with the confocal measurement localized between the two beads at the probe strand binding location. Digital increases (decreases) in fluorescence indicate the binding (unbinding) of a probe strand.

Figure 4

Combined measurement of fluorescence and DNA tether extension. (a) – (b) and (d) – (e) Fluorescence (red, 333 ms per data point) and DNA tether extension (blue, acquired at 66 kHz, boxcar averaged to 3 Hz) are measured simultaneously. The binding (unbinding) of probe strands is indicated by stepwise increases (decreases) in fluorescence and is coincident with angstrom-level stepwise changes in DNA tether extension. (c) and (f) Change in tether extension for many binding and unbinding events for 10 pN (c) and 3 pN (f) tensions. (g) Mean change in tether extension upon binding and unbinding vs. tether tension. Data points at ~3, 5 and 10 pN include only binding events (avoiding confusing unbinding with photobleaching). ~15 pN data point includes only unbinding events (photobleaching does not occur given short bound probe lifetime and insufficient counts of binding were obtained since acquisition was made using a “Wait-and-Yank” technique, see Methods). Standard error bars are smaller than symbol sizes (n = 90, 114, 63, 19, and 14 for 3, 5, 10, 17, and 20 pN respectively). Measurements in (g) and (h) at 3, 5, 10, 17, and 20 pN, are derived from 71, 35, 20, 17, and 16 unique tether molecules respectively. Models assume 9 nt or 7 nt (best fit to data) of the 9 nt probe strand stably bind to the tethered DNA. (h) Semilog plot of duplex lifetime vs. tension (s.e.m, n = 156, 68, 52, 35, and 34 for 3, 5, 10, 17, and 20 pN respectively). Dashed line is linear fit. ~15 and 20 pN data in (g) and (h) acquired with Wait-and-Yank technique.

Figure 5

Proposed reaction diagram illustrating the complete process of probe strand annealing (binding) and melting (unbinding) with complementary tethered DNA. Two free energy contours vs. DNA tether extension are drawn for both the probe strand unbound (blue) and bound (red) states. The initial annealing (binding) and final melting (unbinding) transitions are indicated by the labeled vertical wavy lines. The location of the annealing transition at the extension of the melting transition is assumed given the reversibility of the reaction, although the precise location is not directly measured. Relaxations and excitations of the DNA tether along the reaction contours are indicated by black arrows. The change in DNA tether extension upon annealing is labeled Δx_h. The distance to the melting transition state is labeled Δx₀.