Illuminating Satellite Cells: Light Sheet Fluorescence Microscopy for <scp>3D</scp> Imaging of Murine Skeletal Muscles Damaged by Ex Vivo Forced Eccentric Contraction

In this letter, we put forward the light sheet fluorescence microscopy (LSFM) as a cutting-edge tool for 3D imaging of whole skeletal muscle, focusing on satellite cells (SCs). SCs represent the resident adult muscle stem cells, normally lying quiescent between the sarcolemma of the myofiber and the surrounding basal lamina. They typically express Pax-7 and, when activated following damage, they sequentially express specific myogenic regulatory factors including the myogenic determination factor, MyoD, thus starting differentiation towards multinucleated myofibers to repair injured tissue. The present analysis was performed on an ex vivo model of murine skeletal muscle injured by a forced eccentric contraction in isometric condition. The entire muscles were subjected to a tissue clearing and whole-mount staining process, enabling optical access and specific labeling across the entire intact sample. We performed labeling either with a fluorescent analog of standard hematoxylin and eosin, or with specific immunostaining against Pax-7 and MyoD. This proof of concept study demonstrates the feasibility of whole-muscle imaging with LSFM for the evaluation of the spatial arrangement of resting and activated SCs, overcoming the methodological limits of conventional 2D histology. This innovative experimental pipeline can be useful to test novel therapeutic approaches aimed at enhancing tissue regeneration and other biomedical/clinical applications.