Microscopic viscosity (microviscosity) is akey determinant of diffusion in the cell and defines the rate of biological processes occurring at the nanoscale, including enzyme-driven metabolism and protein folding. Here we establish a rotor-based organelle viscosity imaging (ROVI) methodology that enables real-time quantitative mapping of cell microviscosity. This approach uses environmentsensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartmentspecific microviscosity measurements via fluorescence lifetime imaging. ROVI visualized spatial and temporal dynamics of microviscosity with suborganellar resolution, reporting on a microviscosity difference of nearly an order of magnitude between subcellular compartments. In the mitochondrial matrix, ROVI revealed several striking findings: a broad heterogeneity of microviscosity among individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarization. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes.
J.E.C. was funded by a grant from the Alpha-1 Foundation. M.K. was funded by an Imperial College President’s Ph.D. Scholarship and an EPSRC Doctoral Prize Fellowship. R.G.H. and P.J.B. were funded by A*STAR. E.A. is a UK Dementia Research Institute fellow. S.J.M. was funded by the BLF, the MRC, and the Alpha-1 Foundation. M.K.K. and I.L.D. were funded by the EPSRC in the form of Career Acceleration
Fellowship to MKK (EP/I003983/1).