The fluorescence intensity and lifetime of the 4,4′-difluoro-4-bora-5-(p-oxoalkyl)phenyl-3a,4a-diaza-s-indacene (1) show a strong correlation with the viscosity of the medium due to the viscosity-dependent twisting of the 5-phenyl group, which gives access to the dark nonemissive excited state. We propose a sensitive and versatile method for measuring the local microviscosity in biological systems, based on the determination of the fluorescence lifetime of 1. Fluorescence lifetime imaging (FLIM) performed on live cells incubated with 1 demonstrates the distinct intracellular lifetime of the molecular rotor of 1.6 ± 0.2 ns corresponding to the intracellular viscosity of ca. 140 cP. Time-resolved fluorescence anisotropy of 1 in cells confirms insignificant binding of the fluorophore. The viscosity value obtained in the present study is considerably higher than that of water and of cellular cytoplasm. The high viscosity of intracellular compartments is likely to play an important role in vital intracellular processes, including the rate of diffusion of reactive oxygen species, causing programmed cell destruction.

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 quantum yields, radiative and non-radiative rate constants, fluorescence lifetimes and time-resolved fluorescence anisotropy of the dyes in homogeneous methanol/glycerol solutions of varying viscosity from 0.6 cP 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 cP 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.3