Single-particle tracking of Nucleotide Excision Repair proteins inside living bacteria

Abstract

Single-particle tracking (SPT) combined with Photoactivated Localization Microscopy (sptPALM) provides an opportunity to perform complex molecular biology experiments inside living cells. By tracking the motion of DNA repair proteins in vivo, information can be extracted not only about their diffusion, but also about the kinetics and spatial distribution of DNA binding1-3. From a methodological point of view, a Total Internal Reflection Microscope equipped with a sensitive detector (usually an EM-CCD camera4) is commonly used, allowing detection of individual fluorophores. The signal from individual emitters can be analysed and the position of a given fluorophore established with high accuracy (up to a single nm) by Gaussian fitting. To determine the mobility of each fluorophore, the positions of individual molecules are linked into trajectories over multiple frames using a tracking algorithm5.