Author Description

The visualization of ionizing radiation-induced foci (IRIF) by immunofluorescent microscopy is a proven method of identifying sites of DNA double strand breaks (DNA-DSB). IRIF can be analysed using high-resolution microscopes with appropriate filter sets. In this thesis, I developed a rigorous microscopic approach to IRIF foci image analysis with the development of appropriate algorithms. I then tested the hypothesis that H2AXS139ph IRIF are dose and time responsive and can reflect differences in DNA-DSB repair using normal and DNA-DSB repair-deficient human fibroblasts. The algorithms for automatic foci counting utilized wide-field microscopic deconvolution and multi-pass filtering to determine "true" IRIF over background. Initial H2AXS139ph IRIF can be quantitated using this technique, but it was found that H2AXS139ph IRIF did not predict DNA-DSB repair between the two cell types. Using the methods developed in this thesis, investigators can now pursue dedicated DNA-dsb experiments with an improved "signal to noise" ratio.