Tools to characterize DSB movements in fixed and living cells. (A) Icy bioimaging software (Quantitative Image Analysis Unit, Institut Pasteur) uses the spot detector plug-in and spatial analysis plug-in to assess the clustering of DSB foci in fixed cells (Lagache et al., 2013). The spatial analysis plug-in utilizes Ripley’s K-function to assess the deviance of paired points from total randomness. When sufficient pairs of points congregate within a given radius, the software generates a K-function, which crosses a threshold of statistical significance indicating clustering events (green dots and representative green curve). This allows for analysis of DNA damage clustering using markers that are currently not compatible with live-cell imaging, such as Rad51 or γH2AX. (B) In living cells, DSB movements may be measured as individual foci using the MATLAB plug in @msdanalyzer (Tarantino et al., 2014). MSD values are calculated using the equation MSD = {[x(t + Δt) − x(t)]²}, where x is the position of the DSB focus and t is the time. Particle tracking is shown in the top panels. MSD curves are shown in the bottom panels. Freely diffusing particles display unconfined Brownian motion (top left). The MSD plots the average squared distance that particles travel over increasing time intervals. MSD curves that increases proportionally with time are characteristic of Brownian motion (bottom). Particle movements that exceed diffusion are called directed motions (top middle). MSD curves that increase without bounds as time approaches positive infinity are characteristic of particles with directed motion (bottom). Subdiffusive particles moving within a limited territory are said to display confined Brownian motion (top right). Particles undergoing subdiffusion generate MSD curves that plateau at later time intervals.