Fig. 1

Flowchart representing the image analysis pipeline implemented in our algorithm. The first section of the software (yellow box) is responsible for segmenting the images and extracting the fluorescence intensity emitted by each cell. Beside the segmentation routine, it includes the pre-elaboration phase and its input are the raw images (in 8-bit RGB format). The outputs of this section are i) a pdf file, containing the images at different stages of elaboration, ii) a text file containing each image cell density, computed as number of segmented cells divided by the volume used to prepare the slide and iii) another text file, in which the fluorescence intensities emitted by each segmented cell are reported. This last text file is the input of the second section of the algorithm that is responsible for the analysis of the fluorescence data and for the production of the outputs of our software. It mainly consists in the computation of the statistical modes of the fluorescence distribution (average, standard deviation, CV) and their graphical representation. However it also includes a data analysis step in which the fluorescence intensities are normalized and the cardinality of the populations is equalized