Intravital single-molecule microscopy reveals protein kinetics in live mammalian tissues

Quantifying individual protein behavior in living mammalian tissues remains challenging. Here we introduce intravital single-molecule microscopy (iSiMM), an imaging and analysis framework that combines instant structured illumination microscopy guided tissue navigation with highly inclined and laminated optical sheet illumination. In live mice, iSiMM enables single-emitter detection and tracking of endogenously expressed fluorescently tagged proteins at 30 ms per frame. Trajectory-based modeling quantifies diffusion coefficients and model-defined bound and free fractions, while localization-density reconstructions map molecular organization. We illustrate the approach using the actomyosin cytoskeleton, resolving isoform-specific mobility and responses to physiological stimulation and genetic perturbation. Single-molecule detection in pancreas and skeletal muscle, including after in vivo transfection, further demonstrates feasibility across additional tissues and labeling strategies. iSiMM provides a framework for investigating molecular behavior in the physiological context of living mammalian tissues.