![]() The physiological target of IR is not DNA itself but rather DNA in the context of chromatin, i.e. DSBs are the major threats to the genomic integrity of cells and if insufficiently repaired or misrepaired, DSBs may lead to chromosome breaks, deletions and translocations. Moreover, FocAn was capable of identifying overlapping foci in 3D space, which ensured accurate foci counting even at high DSB density of up to ~ 200 DSB/nucleus.ĭNA double-strand breaks (DSBs) are biologically the most significant lesions produced by ionizing radiation (IR) and other exogenous cytotoxic agents. We found that the data obtained with FocAn agreed well with those obtained with an already available software (FoCo) and manual counting. We validated the FocAn algorithm using fluorescence-labeled γH2AX in two glioblastoma cell lines, irradiated with 2 Gy and given up to 24 h post-irradiation for repair. The FocAn algorithm consists of two parts: nucleus identification and foci detection, each employing specific sequences of auto local thresholding in combination with watershed segmentation techniques. The image-stack processing algorithm implemented in FocAn is capable of automatic 3D recognition of individual cell nuclei and γH2AX foci, as well as evaluation of the total foci number per cell nucleus. To overcome the limitations of 2D foci counting, we present a freely available ImageJ-based plugin (FocAn) for automated 3D analysis of γH2AX foci in z-image stacks acquired by confocal fluorescence microscopy. ![]()
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