Skeletal muscle mitochondrial and lipid droplet content assessed with standardized grid sizes for stereology

Skeletal muscle mitochondrial (Mito) and lipid droplet (Lipid) content are often measured in human translational studies. Stereological point counting allows computing Mito and Lipid volume density (Vd) from micrographs taken with transmission electron microscopes. Former studies are not specific as to the size of individual squares that make up the grids, making reproducibility difficult, particularly when different magnifications are used. Our objective was to determine which size grid would be best at predicting fractional volume efficiently without sacrificing reliability and to test a novel method to reduce sampling bias. Methods: ten subjects underwent vastus lateralis biopsies. Samples were fixed, embedded, and cut longitudinally in ultrathin sections of 60 nm. Twenty micrographs from the intramyofibrillar region were taken per subject at X33,000 magnification. Different grid sizes were superimposed on each micrograph: 1,000 X 1,000 nm, 500 X 500 nm, and 250 X 250 nm. Results: mean Mito and Lipid Vd were not statistically different across grids. Variability was greater when going from 1,000 X 1,000 to 500 X 500 nm grid than from 500 X 500 to 250 X 250 nm grid. Discussion: this study is the first to attempt to standardize grid size while keeping with the conventional stereology principles. This is all in hopes of producing replicable assessments that can be obtained universally across different studies looking at human skeletal muscle mitochondrial and lipid droplet content.