Imaging of Intracellular Viscosity and Membrane Order by New Molecular Rotors Suitable for Phasor Analysis of Fluorescence Lifetime

Abstract

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. We here present two new molecular rotors characterized by a push-pull hyperconjugated structure and which absorb in the blue-green region of the spectrum. These probes display significant emission and lifetime increase by increasing local microviscosity. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to report on environmental properties, such as viscosity, in the cellular context, i.e. where the fluorescent probe concentration cannot be controlled. Remarkably, we demonstrate that our probes allow for efficient intracellular viscosity measurements by means of the phasor approach to fluorescence lifetime imaging, a fit-free method that overcome the main drawbacks of conventional lifetime imaging (1). More specifically, we show that one probe can be used to monitor membrane organization (i.e. the fractions of liquid ordered and disordered phases) and membrane viscosity, whereas the other reports on the local viscosity in plasma membrane, lysosomes, and mitochondria (2). Notably, measurements were carried out in both physiological and non-physiological conditions, in view of correlating dysfunctional states of the cell with viscosity changes in intracellular organelles.1. Digman, M.A., Caiolfa, V.R., Zamai, M., and Gratton, E. (2008) Biophys J 94, L14-16.2. Battisti, A., Panettieri, S., Abbandonato, G., Jacchetti, E., Cardarelli, F., Signore, G., Beltram, F., and Bizzarri, R. (2013) Anal. Bioanal. Chem. 405, 6223-6233.