Agnostic Tracking: Nanoscale, High Bandwidth, 3D Particle Tracking for Biology

The ability to detect biological events at single molecule level provides unique insights in the field of biophysics. Back-focal-plane laser interferometry is a promising technique for single-molecule-scale, 3D position measurements at rates far beyond the capability of video. I present an in-situ calibration method for the back-focal-plane, low-power (non-trapping) laser interferometry. The software-based technique does not rely on any a priori model or calibration knowledge; hence the name Agnostic. The technique is sufficiently fast and non-invasive that the calibration can be performed on the fly, without interrupting or compromising the on-going experiment. The technique can be applied to track 3D, long range motion (up to 100 um) of a broad variety of microscopic biological objects. The spatiotemporal resolution achieved is of the order of a few nanometers and tens of microseconds. Three biological applications enabled by the technique are presented: firstly, a prototype of an oscillating-bead high-bandwidth frequency-response analyzer for biology, based on Agnostic Tracking as implemented in our custom-built 3D Magnetic Force Microscope (3DFM); secondly, a magnetic-force study that revealed a previously-unknown anchoring-dependent nonlinear response of a cellular membrane; last, a rheological study that revealed a novel grouping of motion characteristics of individual vesicles diffusing inside live cytoplasm

Agnostic Particle Tracking for Three-Dimensional Motion of Cellular Granules and Membrane-Tethered Bead Dynamics

The ability to detect biological events at the single-molecule level provides unique biophysical insights. Back-focal-plane laser interferometry is a promising technique for nanoscale three-dimensional position measurements at rates far beyond the capability of standard video. We report an in situ calibration technique for back-focal-plane, low-power (nontrapping) laser interferometry. The technique does not rely on any a priori model or calibration knowledge, hence the name “agnostic”. We apply the technique to track long-range (up to 100 μm) motion of a variety of particles, including magnetic beads, in three-dimensions with high spatiotemporal resolution (∼2 nm, 100 μs). Our tracking of individual unlabeled vesicles revealed a previously unreported grouping of mean-squared displacement curves at short timescales (<10 ms). Also, tracking functionalized magnetic beads attached to a live cell membrane revealed an anchorage-dependent nonlinear response of the membrane. The software-based technique involves injecting small perturbations into the probe position by driving a precalibrated specimen-mounting stage while recording the quadrant photodetector signals. The perturbations and corresponding quadrant photodetector signals are analyzed to extract the calibration parameters. The technique is sufficiently fast and noninvasive that the calibration can be performed on-the-fly without interrupting or compromising high-bandwidth, long-range tracking of a particle