Quantitative imaging of membrane micropolarity in living cells and tissues by spectral phasors analysis

Intracellular micropolarity is essential in several metabolic processes, as it controls membrane permeability, regulating the fluxes of molecules and energy. Here we describe a method for the determination of the micropolarity in living cells using spectral confocal microscopy. The method is based on a phasor analysis of spectrally resolved images of live cells, labelled with the solvatochromic probe Nile Red. An application is provided to extract a polarity profile from the acquired Spectral datasets, which represent the contribution of hyperpolar, polar and non-polar lipids, and to generate a micropolarity map at submicrometric spatial resolution. A metabolic parameter, representing a quantitative index of the fatty acid-triacylglycerol turnover, is also furnished. This method allows a functional profiling of cells and tissues and the detection of metabolic imbalances between lipid storage and usage. • Use of spectral resolved confocal microscopy of Nile Red labelled cells for pixel resolved determination of the membranes micropolarity. • Spectral acquisition increases the specificity and sensitivity of the detection to provide a polarity profile and a metabolic index for fatty acid-TG turnover. • Use of spectral resolved confocal microscopy of Nile Red labelled cells for pixel resolved determination of the membranes micropolarity.