Mapping viscosity in discrete subcellular locations with a BODIPY based fluorescent probe

Numerous 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 benzyl-guanine derivatized boron-dipyrromethene (BODIPY) molecular rotor that is 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 ratiometric confocal microscopy, the latter option greatly extending the accessibility of the technique although limited to relative meassurements. Statement of Significance Local viscosity affects molecular behaviour from diffusion and conformational changes to enzyme kinetics and has important implications for cell and tissue function. Mechanical methods for measurement of viscosity average over large volumes and long times and are thus unsuitable for rapid changes on small scales that are biologically relevant. This paper reports a novel optical fluorescence method using genome edited cells to deliver a viscosity reporter to tightly defined locations inside living cells, from which non-destructive organelle-specific measurements can be repeatedly made. The local viscosity of seven separate organelles in living cultured human cells is shown for the first time, together with the viscosity behaviour of a membraneless organelle as it is induced in cells by stress.