An Optimized Approach to Study Sub-Sarcomere Structure Utilizing Super-Resolution Microscopy with Secondary VHH Nanobodies

The sarcomere is the fundamental contractile unit in skeletal muscle, and the maintenance of its structure is critical for its function. While alterations in sarcomere structure are implicated in many clinical conditions of muscle weakness this area has made limited progress due, in part, to limitations in the ability to robustly detect and measure at sub-sarcomere resolution. Classically the field has relied on approaches including confocal and electron microscopy, but there are technique-specific limitations with respect to resolution, tissue morphology, and protein specific labeling. In this study, our goal was to establish a robust and reproducible method to probe sub-sarcomere protein localization in longitudinal muscle sections. We optimized several steps from tissue preparation to antibody selection and imaging to provide the ability to quantitatively assess spatial distribution of proteins within a single sarcomere. This includes 1) in situ fixation for structural integrity, 2) use of multiple same host-species primary antibodies with Fab fragment antibody blocking to maintain specificity, and 3) the use of super-resolution structured illumination microscopy (SIM) to improve from confocal, along with use of emergent VHH secondary nanobodies to double the resolution. The combination of these methods provides a unique approach to improve visualization of sarcomere structure while simultaneously providing the ability to rigorously probe protein localization. While this study focused on assessment of skeletal muscle structure and provides an important set of tools for analysis of skeletal muscle health in disease and aging, we suggest the methods herein may prove advantageous for research outside of skeletal muscle.