Development and validation of 'AutoRIF': software for the automated analysis of radiation-induced foci

A java-based image analysis system has been developed (AutoRIF) that quantifies the number, size/volume and relative nuclear locations of RIF within 3D nuclear volumes. Our approach identifies nuclei using the dynamic Otsu threshold and RIF by enhanced Laplacian filtering and maximum entropy thresholding steps and, has an application 'batch optimisation' process to ensure reproducible quantification of RIF. AutoRIF was validated by comparing output against manual quantification of the same 2D and 3D image stacks with results showing excellent concordance over a whole range of sample time points (and therefore range of total RIF/nucleus) after low-LET radiation exposure.This high-throughput automated RIF analysis system generates data with greater depth of information and reproducibility than that which can be achieved manually and may contribute toward the standardisation of RIF analysis. In particular, AutoRIF is a powerful tool for studying spatio-temporal relationships of RIF using a range of DNA damage response markers and can be run independently of other software, enabling most personal computers to perform image analysis. Future considerations for AutoRIF will likely include more complex algorithms that enable multiplex analysis for increasing combinations of cellular markers.The use of markers, such as the phosphorylated variant of histone 2A (γ-H2AX), for the detection of radiation-induced double strand breaks (DSBs) is now standard practice for investigating biologically relevant doses of radiation (for a review of the field see Lobrich et al 2010 [1]). For instance, the induction and subsequent repair kinetics of DSB has been determined in a range of cell types upon exposure to varying qualities and radiation doses [2-6]. Additionally, the development of antibodies specific for other relevant proteins in the DNA damage response (DDR) pathway have revolutionised our ability to investigate mechanistic aspects of DSB processing in interphase nuclei. For exam