Targeted fluorescence lifetime probes reveal responsive organelle viscosity and membrane fluidity

Abstract

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer’s Disease and Leigh’s syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging.

Time‐Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments

“…It has previously been used to probe membrane fluidity, [ 17 ] and MD simulations have shown that its chain inserts into the tail region of a DOPC bilayer. [ 6 ] BODIPY‐C12 is readily taken up by HeLa [ 18 ] and SK‐OV‐3 cells [ 17 ] in which it stains LDs, along with other regions. [ 19,20 ] To fully understand BODIPY‐C12 signals in heterogeneous environments, we start our experiments in solution.…”

“…[ 45 ] Finally, the interactions of BODIPY‐C12 was described by the same forcefield parameters as were used previously. [ 6 ] The Verlet cut‐off scheme was employed to generate the pair lists and the electrostatic interactions were calculated using the Particle‐Mesh Ewald algorithm. Both electrostatic and van der Waals interactions were cut off beyond 1.2 nm.…”

“…Another technique, which has received considerable attention lately, is the use of fluorescent molecular rotors (FMRs). [ 4–8 ] FMRs report on viscosity through their fluorescence lifetime; rotation around a central single bond gives access to a non‐radiative relaxation pathway. If the rotation is restricted, such as in high viscosity environments, access to the non‐radiative pathway is diminished and the fluorescence lifetime increases.…”

Imaging mitochondrial matrix viscosity in live cells via fluorescence lifetime imaging (FLIM) of fluorescent molecular rotors

“…The FMR used in this paper is a BODIPY-based FMR with a triphenylphosphonium (TPP+) moiety attached to the rotating unit, referred to as FMR-1. The construction of the calibration plot (based on fluorescence lifetime spectroscopy in glycerol-methanol mixtures of increasing viscosity) has been described elsewhere, 13 as has the synthesis of FMR-1.…”

“…We recently reported one such FMR, targeted to the mitochondria. 13 Changes in mitochondrial viscosity has been linked to a range of diseases, including most notably Alzheimer's 6 and complex I deficiency (leading to Leigh syndrome). 3 The mitochondria is also home to several diffusion-limited enzyme systems which may be disproportionally affected by changes in viscosity.…”