Fig. 1: PECAn is a versatile tool for the analysis of complex 3D images.

a Structure of PECAn image analysis software. Images are initially analyzed in FIJI/ImageJ. In order to preserve three-dimensional information, each Z-plane (1) of each image is analysed sequentially and compared to adjacent Z-planes. The channels of the image—for instance foci/cell death staining (2a), cell patches (2b), and nuclear mask (2c) are split apart and processed independently either by the built-in algorithms or by a WEKA classifier. For instance, the foci/cell death mask (3a) can be used to perform three dimensional segmentation of individual foci and can then be cross referenced against the cell patch ROIs (3b) to yield a density of foci in different regions within the image (4a) and the level of cell death in individual cell patches (4b). Furthermore, cell patch and nuclear stain channels can be used together to identify individual cells in three dimensions (3c). The data are exported as CSV files (5) which can then be uploaded into the R Shiny companion app for statistical analysis and graph generation (6). b PECAn is able to analyse samples derived from diverse tissues and model organisms. PECAn identifies distinct subpopulations of MDCK cells by combining a nuclear GFP marker with seeded-region growing techniques (left). PECAn analyses two distinct genotypes in three dimensions in an adult Drosophila intestine sample (center left). PECAn can segment and analyze individual cell patches in three dimensions in a Drosophila imaginal disc (center right). PECAn identifies density of TUNEL-positive foci in embryonic mouse hearts carrying differently labelled cell patches (right). c PECAn can identify individual cells (left) and individual foci (right) in three dimensions and analyse their position within the tissue, proximity to cell patch borders (yellow lines), or distance to the patch centroid (white lines). Individual cells/foci are colour coded; a red border denotes a cell in the border region of a patch, and a blue border denotes a cell in the centre region of a patch. Scale bars correspond to 50 µM. d Examples of some of the key parameters for which PECAn can assess.