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Microscopic viscosity (microviscosity) is a key 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 environment sensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartment specific microviscosity measurements via fluorescence lifetime imaging (FLIM). ROVI visualised spatial and temporal dynamics of microviscosity with sub-organellar 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 amongst individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarisation. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes.

Molecular rotors are a class of fluorophores that display environmentally-sensitive photophysics. The fluorescence intensities and lifetimes of these molecules strongly depend upon the viscosity of their immediate surroundings, the surrounding “microviscosity”. In this work, molecular rotors are employed to investigate protein aggregation, the assembly of proteins into amyloid. Amyloid materials are a hallmark of several serious human diseases, yet they are also known to have important functional roles in organisms. The physicochemical conditions that lead to protein aggregation are still poorly understood and characterisation of the pathways of assembly is an important goal amongst researchers. It is equally important to study the effects of protein aggregation on living cells, both intrinsic and extrinsic, mediated through interactions with the membrane. In this work, the aggregation of various proteins in vitro and in the cellular environment was studied by measuring fluorescence decays of molecular rotors using time-resolved fluorescence spectroscopy and microscopy. The experimental results revealed mechanistic details of aggregation that were not observed previously with alternative techniques. Moreover, the data provided quantitative measurements of the changes of mechanical properties (i.e. microrheology) of living cells as a result of protein aggregation. As such, the results demonstrate that molecular rotors are a powerful tool for investigating protein aggregation and the supramolecular assembly of proteins in general. Open Access