Semi‑automatic macro ImageJ analysis for collagen and fibrosis from biopsies images in BrightField and polarised light

This Fiji / ImageJ macro aim to help users for calculation of the collagen and fibrosis percentage per area. It works on whole folder with pictures of picro-Sirius red labeled sections from muscle biopsies. Images acquisition must be done twice with RGB images Bright Field and Polarised in the same file (6 channels file).
In order to compare highly variable biopsies, it was decided to retain areas of endomysium only. User is asked to define all areas to be excluded (some sections showed fibrotic perimisium veins, others showed endomysium only). Results are saved in one csv tab including 1) area of the region of interest, 2) the collagen surface (picro-Sirius red marked) and 3) the fibrosis region, with polarized red due to the modelling of collagen fibers.

The macro allows the user to calculate the % of collagen in the slice and the % of collagen fibrosis from each areas (µm²).

Reusable structure

This macro is structured to be reused. The different parts are well identified, therefore it is possible to change basic function and then perform an analysis macro for another staining. Operations are done in a folder with following steps 1) interactive control of the basic parameters 2) opening and treatment of different channels 3) Interactive crop of the area of interest, 4) Interactive selection of the areas to be excluded, 5) analysis, 6) saving of the Images, 7) saving Log and Results files.