Flow chart demonstrating major procedures involved in the automatic focus counting analysis (panel A) and details of the object identification algorithm (panel B) that is used for identification of both foci and nuclei. An image containing two channels (foci channel and nuclear channel, panel A) represents input data. Each channel is initially analysed separately. For focus identification, optional noise filter and normalisation procedures can be applied if necessary. The focus identification results are analysed in conjunction with the collection of objects (nuclei) generated by the nucleus identification procedure. The object identification procedure (panel B) includes Top Hat transformation to minimise background signal and H-Dome based segmentation in combination with intensity threshold. Four parameters are required as user input: the size of the structuring element, dome height, intensity threshold and minimum object size. The object splitting procedure is applied optionally if overlapping objects are present. For nucleus identification, application of Top Hat transformation and H-Dome based segmentation is optional depending on the origin and quality of the image (nuclei channel).