Quantification of sarcomere length distribution in whole muscle frozen sections

Laser diffraction (LD) is a valuable tool for measuring sarcomere length (L-s), a major determinant of muscle function. However, this method relies on few measurements per sample that are often extrapolated to whole muscle properties. Currently it is not possible to measure L-s throughout an entire muscle and determine how L-s varies at this scale. To address this issue, we developed an actuated LD scanner for sampling large numbers of sarcomeres in thick whole muscle longitudinal sections. Sections of high optical quality and fixation were produced from tibialis anterior and extensor digitorum longus muscles of Sprague-Dawley rats (N=6). Scans produced two-dimensional L-s maps, capturing >85% of the muscle area per section. Individual L-s measures generated by automatic LD and bright-field microscopy showed excellent agreement over a large L-s range (ICC>0.93). Two-dimensional maps also revealed prominent regional L-s variations across muscles.