Conventional transmission electron microscopy and image analysis

Samples were sectioned at 60 nm using an ultramicrotome (MTXL, RMC, Tucson, AZ, USA). The sections were double-stained with 2% uranyl acetate for 20 minutes and lead citrate for 10 minutes, then viewed with the aid of transmission electron microscopy operating at 120 kV (Tecnai 12, FEI, Netherlands; H-7600, Hitachi, Tokyo, Japan).

The total number of intermyofibrillar mitochondria per 100 µm² was counted in each group. The concentration of intermyofibrillar mitochondria was also checked in each patient with and without MRC I defects. Every sample was photographed within a randomly selected grid square. The total counts of mitochondria were divided by the total measured area in order to calculate the number of mitochondria per square micron. Finally, the mitochondria density was expressed as the mean number of mitochondria per 100 µm². Statistical analysis was performed using Student's t-test.

The sizes of 50 randomly chosen mitochondria and lipid droplets were measured in each patient using the Image J program (National Institutes of Health). Mitochondria and lipid droplets were circled, and their actual size was calculated using a calibration grid. Values were represented as the mean individual size (in square microns) of mitochondria and lipid droplets. The statistical significance of differences between means was assessed by analysis of variance followed by Student's t-tests. A probability of less than 5% was considered significant (p<0.05). Statistical normalization was used to investigate the relationship between mitochondria and lipid droplets.