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For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e. , intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative process. Stem cell-mediated skeletal muscle regeneration in the mouse model has been classically studied at specific time points by sacrificing the animal and harvesting the muscle tissue for downstream analyses. A method for direct visualization of muscle stem cells to gain real-time information over a long period in a live mammal has been lacking. Here we describe a step-by-step protocol adapted from Webster et al. (2016) to quantitatively measure the behaviors of fluorescently labeled (GFP, EYFP) muscle stem and progenitor cells during homeostasis as well as following muscle injury.

Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice