Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

Abstract

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

High-throughput single-molecule quantification of individual base stacking energies in nucleic acids

“…Our experimental approach provides a fairly direct measurement compared to some other approaches, which included extrapolating stacked/unstacked equilibrium from migration of nicked DNA in urea gels [20], and measuring single-molecule kinetics of on and off rates in end-stacking of DNA origami tubes [22]. One recent paper published during this work used a similar construct design and found a single A|G base stack energy of −2 kcal/mol [54], consistent within error to our measurement. When we compared pairs of our measured base stacking values with previously measured dinucleotide stacks, our energies were larger in all cases by multiples ranging from 1.2 to 2.2.…”

Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

“…Using the high-precision optical setup described in ref. 3 , we measure the transition rates of the 10-bp hairpin by tracking the 10-nm difference of extension between its two states. The time between two transitions is of the order of 10 ms, allowing us to measure N = 10 4 transitions during a typical acquisition time of 100 s and thus to reduce the relative error made on the estimation of the kinetic rates to .…”

“…The extension of the fluctuating probe is acquired in real time using the high-resolution magnetic tweezers setup described in ref. 3 at an acquisition frequency of 1300 Hz with a CMOS camera (UI-3060CP-M, IDS Ueye). The home-made C/C++ acquisition program Xvin can be found online 31 .…”

“…However, the individual binding events of proteins with NAs cannot be directly observed because they generally do not imply sufficiently large extension changes. Spatiotemporal resolution of single-molecule techniques is still making progress 2 and now allows observing the nanometric signals created by oligonucleotide hybridization (≥8 nucleotides (nt)) 3 and helicase stepping 4 . However, the detection of simple binding events that involve Angström-scale extension changes below the second timescale is still out of reach.…”

Strand-switching mechanism of Pif1 helicase induced by its collision with a G-quadruplex embedded in dsDNA

“…They consist in measuring the extensions of single DNA hairpin molecules tethered to a surface on one end and on a magnetic bead on the other end, in function of the force applied to the magnetic bead the DNA hairpin is tethered to. The force is provided by a couple of permanent magnets and is proportional to the magnetic field gradient [21][22][23][24]. The force is calibrated using the fluctuation dissipation theorem and can 2/19 be modified by changing the distance of the magnets to the surface.…”