New Technologies for Tracking Single Molecules in Live Cells

The central dogma of molecular biology encompasses 3 major classes of biomolecules: DNA, RNA and proteins. We are developing and improving methods for tracking each class individually and for tracking different classes simultaneously in order to address our motivating questions. For example, for a nuclear DNA-binding protein, a key question is: what fraction of the protein of interest is DNA-bound and what fraction is freely diffusing? And what are the characteristics of these subpopulations? Single-particle tracking (SPT) is ideally suited to answer this, but conventional SPT of nuclear proteins suffer from at least 4 biases: 1) tracking error; 2) motion-blur bias; 3) defocalization bias; 4) analysis bias. We recently developed an integrated approach to combining spaSPT and Spot-On to overcome these biases. spaSPT integrates previous ideas: photo-activation (sptPALM) is used to image at low densities which minimize tracking errors and stroboscopic excitation is used to avoid motion-blurring (the blurring effect observed when imaging a rapidly moving object). To correct for the fact that freely diffusing proteins move out of the focal plane much faster than DNA-bound proteins (movie below on the right show a simulation illutrating this), we extended and validated a prior kinetic modeling framework - now available as Spot-On, a drag-n-drop website interface - for explicitly accounting for defocalization and precisely infer the different subpopulations. For more details, see also the Tools section.