TEM analyses of subcellular glycogen distribution. (A) Fixed muscle segments were prepared for glycogen visualization by TEM and cut in longitudinal sections. Three fibers were photographed per muscle in a randomized systematic order including 12–16 images from the subsarcolemmal region and 12–16 images from the myofibrillar region. (B) In the myofibrillar images, the volumetric content of intermyofibrillar glycogen (long green arrow) and intramyofibrillar glycogen (short orange arrow) was estimated by point counting (Nielsen et al., 2011; Weibel, 1980). (C) In the subsarcolemmal images, the volume of subsarcolemmal glycogen (blue arrow) per fiber surface area was estimated by point counting and a fiber length measurement (Jensen et al., 2022). (D) After completion of analysis of the first 102 fibers, image-to-image variation showed a mean stereological coefficient of error of 0.14, 0.17, and 0.19 after analysis of 16 images for intermyofibrillar, intramyofibrillar, and subsarcolemmal glycogen, respectively. It was decided for the remaining fibers (n = 172) that 12 photographed images were sufficient per region. (E) Scatterplot of TEM-estimated total glycogen per muscle (mean of three fibers) versus the glycogen concentration determined from a homogenate. Concordance correlation coefficient (CCC) showed a moderate agreement between the two methods. R indicates Person’s correlation coefficient. Dotted red line indicates best linear fit. (F) The relative distribution (in percent) of the three pools to the total volumetric content. Values are mean and 95% confidence interval. n = 60 fibers from 20 rats.