A semiautomated measurement of muscle fiber size using the Imaris software

Abstract

The size and shape of skeletal muscle fibers are affected by various physiological and pathological conditions, such as muscle atrophy, hypertrophy, regeneration, and dystrophies. Hence, muscle fiber cross-sectional area (CSA) is an important determinant of muscle health and plasticity. We adapted the Imaris software to automatically segment muscle fibers based on fluorescent labeling of the plasma membrane and measure muscle fiber CSA. Analysis of muscle cross sections by the Imaris semiautomated and manual approaches demonstrated a similar decrease in CSA of atrophying muscles from fasted mice compared with fed controls. In addition, we previously demonstrated that downregulation of the Ca2+-specific protease calpain-1 attenuates muscle atrophy. Accordingly, both the Imaris semiautomated and manual approaches showed a similar increase in CSA of fibers expressing calpain-1 shRNA compared with adjacent nontransfected fibers in the same muscle cross section. Although both approaches seem valid for measurements of muscle fiber size, the manual marking method is less preferable because it is highly time-consuming, subjective, and limits the number of cells that can be analyzed. The Imaris semiautomated approach is user-friendly, requires little training or optimization, and can be used to efficiently and accurately mark thousands of fibers in a short period. As a novel addition to the commonly used statistics, we also describe statistical tests that quantify the strength of an effect on fiber size, enabling detection of significant differences between skewed distributions that would otherwise not be detected using typical methods.