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 twodimensional 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.
Sarcomeres, the functional units of contraction in striated muscle, are composed of an array of interdigitating protein filaments. Direct interaction between overlapping filaments generates muscular force, which produces animal movement. When filament length is known, sarcomere length successfully predicts potential force, even in whole muscles that contain billions of sarcomere units. Inability to perform in vivo sarcomere measurements with submicrometer resolution is a long-standing challenge in the muscle physiology field and has hampered studies of normal muscle function, adaptation, injury, aging, and disease, particularly in humans. Here, we develop theory and demonstrate the feasibility of to our knowledge a new technique that measures sarcomere length with submicrometer resolution. In this believed novel approach, we examine sarcomere structure by measuring the multiple resonant reflections that are uniquely defined by Fourier decomposition of the sarcomere protein spatial framework. Using a new supercontinuum spectroscopic system, we show close agreement between sarcomere lengths measured by resonant reflection spectroscopy and laser diffraction in an ensemble of 10 distinct muscles.
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