Extended Data Figure 3: Super resolution microscopy allows improved visualization of individual mitochondria.

a, Single confocal microscopy image of an isolated muscle fibre loaded with mitochondrial membrane potential dye, TMRM. PVM, paravascular mitochondria; IFM, intra-fibrillar mitochondria; V, vessel. b, Single stimulated emission depletion (STED) microscopy image of the same fibre as in a. c, The average of a confocal microscopy image stack of a TMRM loaded isolated muscle fibre. d, The average of a STED microscopy image stack of the same fibre as in c. For c and d, image volume was 24.30 × 27.47 × 1.75 µm in x, y and z, and images shown are the average of 9 images taken with 219 nm z-steps. Confocal images were acquired before STED images at each image depth. All images were acquired with x and y pixel sizes of 30 nm. Scale bars, 2 μm. As most muscle mitochondria are larger than 300 nm, the improved resolution with STED is primarily noted by the increased clarity of the spaces between individual mitochondria, particularly the PVM. Images are representative of 13 fibres from 3 mice.