Computational Image Processing and Analysis of Heat-induced .GAMMA.H2AX Foci

The counting and analysis of γH2AX foci is a widely used technique because these foci are considered to be a reliable indicator for the presence of DNA double-strand breaks (DSBs). The most frequently used method to count foci is to examine images and count by eye, but such an approach may not be the most accurate method to use. An alternative method to manual counting is proposed here, and this method is faster, easier, more objective, and can eliminate artifacts which can result from human handling. A dual-step algorithm is described which uses computational methods and consists of two distinct processes : (i) a 5-filter image filtering and processing step, and (ii) a foci counting/analyzing step. The benefits of this 2 stage computational analysis are primarily in the fact that the filtering process compensates for common preparation errors, as well as for image blurring and foci overlap. This method increases the sharpness of the foci edges and converts a 3D focus distance to an optical density, which can then be measured by the counting software. This allows researchers to compare the results of foci counts with increased reliability and objectivity. The features of this method also permit batch microscope images to be processed automatically. This system has a wide potential range of applications, including foci analysis from different types of immunofluorescence staining, and could form the basis for a standardized computational counting method which could eliminate counting subjectivity and provide an increase in counting reliability.