Fluorescence Anisotropy of Molecular Rotors

Abstract

We present polarization-resolved fluorescence measurements of fluorescent molecular rotors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), 9-(2,2-dicyanovinyl)julolidine (DCVJ), and a meso-substituted boron dipyrromethene (BODIPY-C(12)). The photophysical properties of these molecules are highly dependent on the viscosity of the surrounding solvent. The relationship between their quantum yields and the viscosity of the surrounding medium is given by an equation first described and presented by Förster and Hoffmann and can be used to determine the microviscosity of the environment around a fluorophore. Herein we evaluate the applicability of molecular rotors as probes of apparent viscosity on a microscopic scale based on their viscosity dependent fluorescence depolarization. We develop a theoretical framework, combining the Förster-Hoffmann equation with the Perrin equation and compare the dynamic ranges and usable working regimes for these dyes in terms of utilising fluorescence anisotropy as a measure of viscosity. We present polarization-resolved fluorescence spectra and steady-state fluorescence anisotropy imaging data for measurements of intracellular viscosity. We find that the dynamic range for fluorescence anisotropy for CCVJ and DCVJ is significantly lower than that of BODIPY-C(12) in the viscosity range 0.6<η<600 cP. Moreover, using steady-state anisotropy measurements to probe microviscosity in the low (<3 cP) viscosity regime, the molecular rotors can offer a better dynamic range in anisotropy compared with a rigid dye as a probe of microviscosity, and a higher total working dynamic range in terms of viscosity.

Molecular rheology of neuronal membranes explored using a molecular rotor: Implications for receptor function

“…For this purpose, we generated a calibration curve (see Fig. 2b) by plotting the variation in steady state fluorescence anisotropy of BODIPY-C12 as a function of viscosity using methanol-glycerol mixtures of varying composition (viscosity values of glycerol-methanol mixtures were taken from the literature) (Levitt et al, 2009(Levitt et al, , 2011. The figure shows that at the higher end of the viscosity range, the fluorescence anisotropy approaches ro, i.e., the limiting (fundamental) anisotropy of the BODIPY in the absence of any depolarizing processes such as rotational diffusion (Karolin et al, 1994).…”

“…The viscosity values of glycerol-methanol mixtures were taken from previous literature (Levitt et al, 2009(Levitt et al, , 2011.…”

“…BODIPY is a popular fluorescent probe and is characterized by high extinction coefficient, quantum yield and photostability (Johnson et al, 1991). Fluorescent molecular rotors are interesting reporter molecules that are generally characterized by an excited state process that is dependent on an intramolecular rotational motion Levitt et al, 2011;Kuimova, 2012). A fluorescent molecular rotor typically consists of an electron donor moiety covalently linked to an electron acceptor unit via a spacer that conjugates the donor and the acceptor units, and facilitates electron movement between the two.…”

Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes

“…This curve can be used as a calibration curve for the determination of the local viscosity of the BODIPY-C 12 labels in other measurements. It has previously been postulated that BODIPY-C 12 labels membranes of intracellular organelles ( 47 ). Because BODIPY dyes in general are very good labels for LDs ( 48 ), we however assumed that BODIPY-C 12 with its additional long alkyl chain would accumulate in LDs.…”

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.…”