TMRM staining of mitochondria in WT and DMD ^mdx muscle fibers. (A) Confocal images of TMRM fluorescence in a WT and in a DMD^mdx fiber at two distinct magnifications. Corresponding transmitted light images are shown on top. Black and white frames at the bottom correspond to the binary images obtained after thresholding the above images. (B) Distribution of the values for the area of separate mitochondrial objects in the two groups, up to 20 µm². Each symbol corresponds to the percentage of identified objects exhibiting a given surface, with an increment of 0.2 µm², from all WT and DMD^mdx muscle fibers. (C) Individual and corresponding mean (±SD) values for the area of mitochondrial objects smaller than 5 µm² (data are from 26 fibers from four WT animals and 28 fibers from five DMD^mdx animals. P = 0.38, nested t test, 95% CI for the difference between mean values: −0.29 to 0.13). Each bar and superimposed data points correspond to data from a same animal (arrows point to the series of large events detected in the same groups of fibers in E). (D) Distribution of the values for the area of mitochondrial objects >20 µm² in the two groups. Each symbol corresponds to the percentage of identified objects exhibiting a given surface, with an increment of 50 µm², from all WT and DMD^mdx muscle fibers. (E) Individual values for the area of all mitochondrial objects >20 µm² in fibers from each animal (each column of data points corresponds to fibers from the same animal as in C; each symbol corresponds to an individual area in a given fiber). The red inset shows an example of binary image from a large object detected in a DMD^mdx fiber (190 µm², corresponding data point also shown in red in the graph). 95% CI, 95% confidence interval.