Fig. 3

Gray-level co-occurrence matrix (GLCM) for the quantification of lysosome intracellular distribution. Representative images of the CD68 labeling showing clustered lysosomes at 48 h (a, a′) vs. spread lysosomes at 7 days (b, b′) after pMCAo (yellow outline obtained tracing over CD11b, asterisks indicate nuclei). Scale bars = 10 μm (a) and 1 μm (a′). Testing images on four different gray levels (GL) representing clusters of pixels with same GL (test 1) vs. pixels with different GL (test 2, c). With gray-level co-occurrence matrix analysis, test 1 showed higher angular second moment (ASM) and inverse difference moment (IDM) and lower entropy than test 2. Testing images from 48 h and 7 days pMCAo mice showing the CD68-positive pixels in a spectrum modality (c′). GLCM showed higher pixel homogeneity at 48 h, when clustered pixels were found (c′, right graphs). GLCM on CD68-positive pixels showing higher ASM and IDM (d, e) and lower entropy (f) at 48 h than 7 days. Morphological parameters like circularity (g) and solidity (h) did not differ between time points, thus ruling out any contribution of cell morphology to pixel distribution analysis. Individual cell values with line at mean (n = 20 cells, full points) and mean values per mouse (n = 4 mice, empty points). Normal distribution by Kolmorgov-Smirnov analysis, t test, ***p < 0.001