File:Islet-Formation-during-the-Neonatal-Development-in-Mice-pone.0007739.s005.ogv

English: Screen capture of the automated macro image processing with ImageJ. An ImageJ macro, i.e. a script of instructions written for execution in ImageJ, contains the instructions for the analysis of each sample. An investigator initiates analysis by running the macro, while a virtual slice image is open (3 s). ImageJ starts parsing the macro line by line, creating a duplicate of each virtual slice sample for image processing (5 s). Duplicating the original virtual slice allows the background subtraction parameter to be optimized without having to reload the image. A threshold is automatically chosen to isolate fluorescing particles for quantification (7 s). Selected particles are reduced to a binary black and white representation (9 s). Islets and small beta-cell clusters, represented in black, are quantified. The results window on the bottom right corner displays the parameters of each particle and stores these measurements as a spreadsheet (12–26 s). The summary window appears and the window entitled “beta-1” closes when particle analysis is completed. A single virtual slice image is analyzed in under 30 seconds. Multiple images can be analyzed by embedding this image processing and analysis script into a loop syntax supported by the ImageJ macro language. The loop opens all the files in a given directory, analyzes them, and outputs results into a new location. Virtual slice background levels must be subtracted and artifacts removed prior to multiple image analysis to ensure consistency and accuracy.