Quantifying Extracellular Matrix Patterns

All stained sections were scanned with a Slide Scanner (Hamamatsu Photonics K.K., Herrsching, Germany) at 20× magnification (Figure 1e). PSR fluorescent images (PSR-fluo) were generated with Zeiss LSM 780 CLSM confocal microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany), λ_ex 561 nm/λ_em 566/670 nm at 40× magnification [38 (link)]. COL1A1, Elastin scans and PSR-fluo images were analyzed with TWOMBLI, an ImageJ/Fiji [39 (link)] plugin to quantify patterns in ECM [40 (link)]. Before analyzing the COL1A1 and Elastin, the Vector^® NovaRED™ color was isolated from the images using a color deconvolution plugin [41 (link)]. The images with only Vector^® NovaRED™ color were subsequently used for the analysis.
TWOMBLI was used to determine the number of fibers, end points, branching points, total fiber length and alignment, lacunarity (number and size of gaps in the matrix) and high-density matrix proportion (measure for compactness of matrix).