Fig. 1

The ImageJ-based interface of the Focinator offers options to adapt the evaluation parameters to distinct image characteristics. Figure 1 shows ImageJ with the Focinator macro installed as start-up macro after opening a multi-channel image. This microscope image with the file format ZVI 16-bit includes three fluorescence channels. The main window of the Focinator is implemented into the ImageJ window. It consists of a menu (2), buttons (1) and Focinator Options (3 and 4). The Focinator Options windows offer several preferences for the user to adapt the macro’s behavior to individual requirements. Picture Settings: First step is to tell the macro, the input folder and if there is a multi-channel image or more single pictures will be opened. In the second step you choose in which channel the foci have to be counted and where the ROIs should be selected. In our example, the γ-H2.AX foci are in channel number 2 (on top after opening the image). The macro will use the setting “1st foci channel = front channel” for all pictures automatically. If no second foci channel is used the setting should be changed to “inactive”. ROI Settings (3): Depending on image quality, size and magnification, it is recommended to set the threshold and the size filters for ROIs. Alternatively, the choice of automated thresholding is possible. It is possible to exclude objects that are partially outside of the image. If there are objects to exclude because they are not circular enough or too small, it is possible to exclude them via circularity filters or size filters. “Use fill holes” should be activated, if the ROI selection left holes in the cells. Overlapping ROIs (cells, nuclei) might be separated by choosing “watershed”. Regarding the batch mode “check selection” offers the possibility of stopping during the selection process. “Invert images” should be checked when working with images with light background. For the automated batch (4) mode, output directories need to be chosen to save the results. An important step of evaluation is to choose the right noise level. Noise level values can be set independently in multi-channel analysis to exclude background artifacts. By defining the cut off, foci with intensities below a certain value are deleted, which excludes background noise. The value for area correction is dependent on the mean size of the analyzed nuclei. The factor corrects the foci number divided by the individual area of each nucleus. The usage of the percentile option enables the user to delete the outliers, such as cells with false γ-H2.AX foci induced by replication. Colocalization analyses are also possible. This option compares the localization of two foci in two different channels with a selectable tolerance