Gamma-H2AX foci counting: image processing and control software for high-content screening

Abstract

Phosphorylation of the chromatin protein H2AX (forming γH2AX) is implicated in the repair of DNA double strand breaks (DSB's); a large number of H2AX molecules become phosphorylated at the sites of DSB's. Fluorescent staining of the cell nuclei for γH2AX, via an antibody, visualises the formation of these foci, allowing the quantification of DNA DSB's and forming the basis for a sensitive biological dosimeter of ionising radiation.We describe an automated fluorescence microscopy system, including automated image processing, to count γH2AX foci. The image processing is performed by a Hough transform based algorithm, CHARM, which has wide applicability for the detection and analysis of cells and cell colonies. This algorithm and its applications for cell nucleus and foci detection will be described. The system also relies heavily on robust control software, written using multi-threaded cbased modules in LabWindows/CVI that adapt to the timing requirements of a particular experiment for optimised slide/plate scanning and mosaicing, making use of modern multi-core processors. The system forms the basis of a general purpose high-content screening platform with wide ranging applications in live and fixed cell imaging and tissue micro arrays, that in future, can incorporate spectrally and time-resolved information.

Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

“…The γH2AX and Rad51 foci formation was investigated according to Barber et al [18]. For immunofluorescence staining for γH2AX and Rad51, cells were fixed in 1% paraformaldehyde for 10 min at room temperature followed by 70% ethanol for 10 min at room temperature.…”

γH2AX foci as a measure of DNA damage: A computational approach to automatic analysis

“…Some of these computational approaches are based on the use of the freely available or commercial image analysis software such as ImageJ [32], NIH Image [33] and its Windows counterpart Scion Image [34], MetaMorph (Molecular Devices Inc, US), HistoLab (Microvision Instruments, France), Image Pro (Media Cybernetics Inc, US), AutoQuantX (Meyer Instruments Inc, US), MetaCyte (MetaSystems GmbH, Germany) These approaches invoke computational algorithms at various stages of analysis, starting from image segmentation and focus identification and ending with calculation of numerous quantitative focus parameters, often including writing of custom macros or plug-ins for execution of specific tasks [35–42]. Other approaches rely on custom software developed for dedicated purpose of focus counting analysis such as for example, a standalone FociCounter [43, 44] or focus counting incorporated into automated high throughput image acquisition and processing platform [43, 45]. Computational approaches substantially increase the productivity of the focus counting analysis and, as claimed by some researchers, are free of the unavoidable operator subjectivity and potential bias of the manual counting analysis.…”

Radiosensitization of glioblastoma cells using a histone deacetylase inhibitor (SAHA) comparing carbon ions with X-rays

“…The time duration of imaging all dishes was small, so degradation of the mercury arc lamp, or bleaching of the YAG:Ce scintillator over this time period can be assumed to be negligible. The images were ultimately analysed using a custom made software to measure the area and the peak intensity of the nuclei (Barber et al 2007). …”