Molecular Rotor Measures Viscosity of Live Cells via Fluorescence Lifetime Imaging

Abstract

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.

Singlet Oxygen in a Cell: Spatially Dependent Lifetimes and Quenching Rate Constants

“…47,54 As the solution viscosity is increased, however, the magnitude of the bimolecular quenching rate constant decreases indicating that the process of singlet oxygen deactivation is now determined by the rate at which singlet oxygen and NaN 3 encounter each other (i.e., the diffusion-controlled limit). Considering that the apparent intracellular diffusion coefficient for a small molecule such as oxygen can be comparatively small, [25][26][27][28] and that subcellular domains can have apparent viscosities that are comparatively large, 29 the data shown in Figure 5 provide the necessary framework to interpret data recorded upon the quenching of intracellular singlet oxygen by NaN 3 . We note here that while the intracellular matrix is intrinsically heterogeneous, it is expected that, on a microscopic scale, homogeneous domains will exist.…”

“…The comparatively small values of intracellular diffusion coefficients are consistent with independent data that point to subcellular domains that can be quite viscous. 29 For the present study, we set out to record singlet oxygen data that reflected the heterogeneity of a single cell. On one hand, we were interested in seeing if we could obtain spatially-dependent lifetimes of singlet oxygen that might reflect the unique chemical composition of a given domain.…”

A Molecular Rotor that Measures Dynamic Changes of Lipid Bilayer Viscosity Caused by Oxidative Stress

“…Consequently, a wide range of fluorescent probes suitable for probing multiple properties of lipid membranes was developed,13 including probes for sensing membrane potential and fluidity,14 for detecting lipid order in the outer lipid leaflet of the lipid bilayer15 and for sensing changes in the membrane during apoptosis 16. In this work, we utilized BODIPY‐C 10 17, 18 (Figure 1), a fluorophore that belongs to a group of dyes termed ‘molecular rotors’ that have viscosity‐dependent fluorescence quantum yields, lifetimes,19, 20 and depolarization 21, 22. When combined with fluorescence lifetime imaging microscopy (FLIM), molecular rotors can be used to obtain spatially resolved viscosity maps of microscopic objects,17, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 as well as to observe dynamic change in viscosity during relevant processes of interest 37, 39, 41, 42.…”

Dual mode quantitative imaging of microscopic viscosity using a conjugated porphyrin dimer

“…It is now well established that quantitative imaging of viscosity requires either ratiometric detection or fluorescence lifetime measurements, in order to rule out the effect of the concentration of the probe on the observed signal. [16][17][18][19] Consequently, both types of molecular rotors, using either ratiometric or lifetime detection, have been reported and used for imaging in a variety of systems, from live cells to lipid membranes to atmospheric aerosols. 2,[16][17][18]20 Here we report a red emitting molecular rotor 1, Figure 1a, previously synthesized by Balaz et.…”