01-08-2012, 03:00 PM
2D Fluorescent Image Analysis for Automation Approach to Quantifying DNA Damages using γ-H2AX
DNA DSBs quantification.doc (Size: 579 KB / Downloads: 40)
ABSTRACT :
Damage to DNA and especially double strand breaks(DSBs) play a critical role both in the emergence and treatment of cancer. A single double strand break may be enough to bring about alterations in the genome that will eventually lead to cancer or cell death. When a double strand break is introduced in the DNA the cell is signalling this by phosphorylating the histone protein H2AX (forming γ/gamma H2AX). It 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.
But due to time consuming nature of the assay it is not suitable for the study of large number of samples or the get the quick results.
INTRODUCTION:
Counting of γ-H2AX foci is now widely used for the quantitative evaluation of the induction and repair of DSBs by various cytotoxic agents, including ionizing radiation (9–11). In addition to γ-H2AX, several other proteins involved in the cellular responses to DNA damage form nuclear foci that are quantified in a similar manner.
Despite the wide use and importance of focus analysis in experiments evaluating cellular responses to DNA damage, focus quantification is frequently based on manual counting even when used to obtain information regarding numbers of DSBs per cell or to derive conclusions on underlying mechanisms. Manual focus scoring has a number of disadvantages.
Image capture:
Olympus BX-51 fluorescence microscope with CCD-camera is used to acquire the images of γH2AX. For each patient, 4 different repair points, for each repair points 40 cells to capture (for 4 repair points total 160 cells), each cell contains ~5 slices (total 800 images).
Image analysis:
Overlapping of 5 slices or images, adjusting the threshold so that the foci appears brighter and background get reduced, counting the number of foci in the cell.
CONCLUSION AND FUTURE WORK:
A fully automated image analyzing system forms the basis for a high-content screening platform and a system aimed at the analysis of γH2AX foci has been described. The general purpose acquisition software aids experiment design, but in-house development allows easy optimisation and adaptation for specific tasks as well as time and cost-efficient development of working systems. Automated processing of this type offers objective counting and the collection of additional statistics on size, shape, position and distance to the nearest neighbour.