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

iSiMM tracks endogenously expressed fluorescently tagged proteins in live mice at 30 ms per frame, enabling single-emitter detection and trajectory-based kinetic modeling.

• Why it matters: Measuring individual protein kinetics in intact mammalian tissues is difficult, limiting how well molecular behavior can be quantified in physiological context.
• What they did: The iSiMM framework combines tissue navigation with instant structured illumination microscopy and highly inclined laminated optical sheet illumination, then uses trajectory-based modeling to estimate diffusion coefficients and bound/free fractions.
• The result: In live mice, iSiMM resolves isoform-specific mobility and responses to physiological stimulation and genetic perturbation in the actomyosin cytoskeleton, and detects single molecules in pancreas and skeletal muscle after in vivo transfection.

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