AbstractNumerous cellular processes, including enzyme behaviour, signalling, and protein folding and transport are highly influenced by the local microviscosity environment within living cells. Molecular rotors are fluorescent molecules that respond to the viscosity of their environment through changes in both the intensity and lifetime of their fluorescence. We have synthesised a novel boron-dipyrrin (BODIPY) molecular rotor that is also a substrate for the SNAP-tag targeting system (named BG-BODIPY), allowing us to target the rotor to discrete locations within the living cell. We demonstrate that BG-BODIPY reports viscosity, and that this can be measured either through fluorescence lifetime or intensity ratiometric measurements. The relative microviscosities within the ER, Golgi, mitochondrial matrix, peroxisomes, lysosomes, cytoplasm, and nucleoplasm were significantly different. Additionally, this approach permitted fluorescence lifetime imaging microscopy (FLIM) to determine the absolute viscosity within both mitochondria and stress granules, showcasing BG-BODIPY’s usefulness in studying both membrane bound and membraneless organelles. These results highlight targeted BG-BODIPY’s broad usefulness for making measurements of cellular viscosity both with FLIM and conventional confocal microscopy, the latter option greatly extending the accessibility of the technique.
Membrane-Bound Molecular Rotors Measure Viscosity in Live Cells via Fluorescence Lifetime Imaging
