Fluorescence lifetime imaging microscopy of flexible and rigid dyes probes the biophysical properties of synthetic and biological membranes

Sensing of the biophysical properties of membranes using molecular reporters has recently regained wide-spread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging (FLIM) of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile Red and NBD, the viscosity sensor Bodipy C12, the flipper dye FliptR as well as the dyes DiO, Bodipy C16, lissamine-rhodamine, Atto647 which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with the well-known environmental reporters, with Nile Red and Bodipy C12 being specific reporters of medium hydration and viscosity, respectively. We show that some widely used ordinary dyes with no previous report of sensing capabilities can exhibit competing performance compared to highly sensitive commercially available or custom-based solvatochromic, molecular rotors or flipper in a wide range of biophysics experiments. Compared to other methods, FLIM is a minimally invasive and non-destructive method with optical resolution. It enables biophysical mapping at steady state or assessment of the changes induced by membrane-active molecules at subcellular level in both synthetic and biological membranes when intensity measurements fail to do so. The results have important consequences for the specific choice of the sensor and take into consideration factors such as probe sensitivity, response to environmental changes, ease and speed of data analysis and the probe’s intracellular distribution, as well as potential side effects induced by labelling and imaging.