Precision Surface-Coupled Optical-Trapping Assay with One-Basepair Resolution

Abstract

The most commonly used optical-trapping assays are coupled to surfaces, yet such assays lack atomic-scale ( approximately 0.1 nm) spatial resolution due to drift between the surface and trap. We used active stabilization techniques to minimize surface motion to 0.1 nm in three dimensions and decrease multiple types of trap laser noise (pointing, intensity, mode, and polarization). As a result, we achieved nearly the thermal limit (<0.05 nm) of bead detection over a broad range of trap stiffness (k(T) = 0.05-0.5 pN/nm) and frequency (Deltaf = 0.03-100 Hz). We next demonstrated sensitivity to one-basepair (0.34-nm) steps along DNA in a surface-coupled assay at moderate force (6 pN). Moreover, basepair stability was achieved immediately after substantial (3.4 pN) changes in force. Active intensity stabilization also led to enhanced force precision ( approximately 0.01%) that resolved 0.1-pN force-induced changes in DNA hairpin unfolding dynamics. This work brings the benefit of atomic-scale resolution, currently limited to dual-beam trapping assays, along with enhanced force precision to the widely used, surface-coupled optical-trapping assay.

Practical axial optical trapping

“…7,25 Located between the AOD and the microscope objective (Nikon CFI × 100, oil immersion, NA 1.25) is a telescope arrangement that expands the beam diameter by a factor of three, ensuring that the back pupil of the microscope objective is overfilled, as required for strong trapping. Beyond the objective, the beam is directed onto a quadrant photo-diode (QPD) (Phresh Photonics SiQu50-M) located in a plane conjugate to the back focal plane of the microscope condenser lens, optimized for axial detection.…”

“…[17][18][19][20][21][22][23][24] Optical traps have proven particularly effective in elucidating the effects of tension on the behavior of single molecules tethered between a surface and a trapped bead 6,7,25 or between two trapped beads. 11,12,15,19,26,27 The dumbbell configuration, in which the molecule of interest is tethered between two trapped beads, has a simple readout and decouples the molecule's extension from microscope stage noise.…”

“…In fact, it has been shown too that surface attachment schemes can be implemented in very-low-noise setups. 25,[35][36][37] In most surface tethered applications to-date, tension has been applied to surface-tethered molecules by moving the stage perpendicular to the direction of beam propagation. Such a geometry yields a simple conversion of the scattering light intensity to force 38 for relatively long tethers.…”

Single DNA molecule detection in an optical trap using surface-enhanced Raman scattering

“…19,21 This value would change proportionally to the number of molecules being extended. In the sample set, the persistence length values ranged between 47-57 nm which is within reasonable error of the well-established value for a single DNA molecule.…”

Quantifying and pinpointing sources of noise in optical tweezers experiments

“…Much effort has been put into minimizing noise, for instance entire setups have been covered to eliminate pressure fluctuations; the equipment is most often placed on an optical table and sometimes even on a foundation which is separated from the rest of the building. A successful way to eliminate drift is by using a laser beam parallel to the trapping laser to track the motion of a feducial marker which is attached in proximity to the handle of interest and thus subject to a similar drift [10]. This setup, however, requires the use of at least two laser beams and two independent detection systems.…”