A novel optical imaging system for investigating sarcomere dynamics in single skeletal muscle fibers

The protein substructure of skeletal muscle fibers forms a diffraction grating with repeating units, termed 'sarcomeres'. A laser scanning system is described that maps the lengths of sarcomeres (SL) and the widths of the first-order diffraction lines (DLW) of permeabilized single fibers in real-time. The apparatus translates a laser beam (lambda = 670 nm and w(0) = similar to 75 mu m) along the length of a fiber segment through 20 contiguous regions per sweep at 500 sweeps/s. The fiber segments (similar to 1 mm long) were obtained from vastus lateralis muscles of humans by needle biopsy. During both passive stretches and maximum fixed-end activations, the mappings of SL and DLW of the fibers were extracted from the diffraction spectra. Heterogeneity of SLs was evaluated by computing the standard deviation (sigma(SL)) of the 20 SLs measured during a single sweep. Compared with the sigma(SL) before a passive stretch, the increase of 5 +/- 0.5% in sigma(SL) after the passive stretch, indicated differences in passive length-tension relationships along the fiber. In contrast, no change, similar to 0.5 +/- 0.1%, was observed in DLW. Within 10s after the fiber was returned to its initial length, the shape of the SL profile returned close to pre-stretch conditions (sigma(SL) = 1 +/- 0.2%). Following maximum Ca2+ - activation of the fiber, the heterogeneity of the steady state SLs increased greatly (DLW up by similar to 300% and sigma(SL) up by similar to 100%). The scanning system provided high resolution tracking of sarcomere behavior single muscle fibers. Potential applications are for studies of the mechanisms of muscle fiber injury and injury propagation.