Membrane-Bound Molecular Rotors Measure Viscosity in Live Cells via Fluorescence Lifetime Imaging^†

We report intracellular fluorescence lifetime imaging (FLIM) and fluorescence anisotropy measurements of two meso-substituted fluorophores based on the boron-dipyrrin (BODIPY) structure. Both dyes incorporate hydrophobic groups, which render them membrane-soluble. We have obtained values for quantum yields, radiative and nonradiative rate constants, fluorescence lifetimes, and time-resolved anisotropy data for the dyes in homogeneous methanol/glycerol solutions of varying viscosities from 0.6 to 950 cP. We find that the fluorescence lifetimes and rotational correlation times for both dyes increase with increasing viscosity, as predicted by theory. These molecules can thus serve as fluorescent molecular rotors to report on local microviscosity, including that in live cells. The dyes are readily taken up by cells as imaged using confocal fluorescence microscopy. Using FLIM, we have detected two distinct fluorescence lifetime populations for both dyes in live SK-OV-3 human ovarian carcinoma cells, corresponding to apparent viscosity values of 160 +/- 20 and 260 +/- 40 cP, each found in distinct intracellular domains. In both cellular domains, independent of the fluorophore used, the viscosity values significantly exceed that expected for the aqueous phase of cellular cytoplasm, suggesting slower diffusion and reaction rates in this hydrophobic microenvironment. FLIM measurements were complemented with time-resolved fluorescence anisotropy measurements, which confirm the high viscosity values in the immediate environment of both rotors. The present study highlights the power of FLIM to map heterogeneous microenvironments of complex biological systems and also the use of fluorescent molecular rotors as microviscosity sensors.