Image Correlation Spectroscopy is a Robust Tool to Quantify Cellular DNA Damage Response

The DNA Damage response (DDR) is both essential and highly complex. Evaluating the DDR is a critical aspect of cell biology. Counting DNA damage foci is one of the most common approaches to study the DDR. Yet, quantification of protein foci suffers from experimental limitations, subjectivity of analysis and is restricted to a handful of the hundreds of DDR proteins. Here we apply image correlation spectroscopy (ICS) to quantify the local clustering at sites of DNA damage directly. We found that ICS outperformed foci counting of traditional DDR markers and enabled quantification of other markers without the complex labeling procedures that are otherwise required. ICS analysis also provided insight into DDR protein recruitment that was previously undetectable. Further expansion incorporating analysis to cell cycle classification demonstrates a rapid, non-biased approach to fully study the DNA damage response within cells. ICS analysis presents an objective, quantitative image analysis technique to study the DNA damage response in unaltered cells that we expect will significantly enhance quantitative DNA damage response research.