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

AbstractThe 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 100μm) motion of a variety of particles, including magnetic beads, in three-dimensions with high spatiotemporal resolution (∼2nm, 100μs). Our tracking of individual unlabeled vesicles revealed a previously unreported grouping of mean-squared displacement curves at short timescales (