Measuring Skeletal Muscle Mitochondrial Architecture in 3D Using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM).

Recent advances in volume electron microscopy have permitted unprecedented visualizations and in-depth quantitative analyses of mitochondrial network structure in skeletal muscle. Nanoscale, 3D analyses of skeletal muscle mitochondria have provided novel insights into how mitochondria interact among themselves and with other skeletal muscle cell organelles, such as the sarcoplasmic reticulum and the sarcomere. The identification of mitochondria within volume electron microscopy datasets and the downstream analyses of segmented structures remain two major bottlenecks in the analysis of 3D mitochondrial network structure at the nanoscale. Here, we provide a step-by-step guide on how to use machine learning programs to segment skeletal muscle mitochondria from volume electron microscopy datasets, followed by instructions on several types of 3D analyses of individual mitochondria and mitochondrial network structure that can be performed using ImageJ. We highlight key quality-control steps to ensure precise and accurate segmentation and analyses and to enhance reproducibility across groups.