Live cell imaging can monitor biological processes in time and space by providing quantitative measurements of cell behavior on a single-cell basis and in live conditions. However the illumination required to visualize fluorescently tagged endogenous proteins often perturbs cellular physiology, a problem particularly acute for yeast cells that are small, highly photosensitive and with scarce protein content. Analyzing the activation of the DNA damage response (DDR) in various yeast mutants or growth conditions, as well as its consequences for cell cycle progression and cell viability over extended periods of time therefore requires a special microscopy setup that does not by itself create DNA damage or perturb cell growth. Here, we provide a quick guide, strains and advice for imaging the DDR in S. cerevisiae for extended time (3-12 h) using spinning-disk confocal microscopy in conditions of limited photobleaching and photodamage. DDR is a conserved mechanism that allows the cell to respond to various stresses, especially those altering DNA integrity or topology. Acquiring time-lapse images of the DDR at high temporal and spatial resolution is of great interest, in particular when studying the effects of mutations or drugs which compromise genomic stability and cell cycle progression.
Keywords: DNA damage response; Microscopy; Phototoxicity; Rad52-GFP; Recombination foci; S. cerevisiae; Spinning disk microscopy; Yeast; Yokogama CSU-X1; mCherry-Tub1.