Multicomponent Analysis of Phasor Plot to Decipher Changes in Metabolic Trajectory of Biological Systems

Abstract

Recent developments of phasor approach to Fluorescence Lifetime Imaging (FLIM) in cells undergoing oxidative stress or cultured in oleic acid enriched media and in mice liver sections having high fat diet, have shown presence of a third autofluorescent component indicative of lipid droplets besides the phasor components from free and enzyme bound NADH in the 400-450 nm emission wavelength. This third component changes the position and shape of the phasor distribution away from the metabolic trajectory, defined by the line joining the phasor position of free and enzyme bound NADH, and makes the study of changes in metabolism using the ratio of free to bound NADH difficult. However, the phasor rule of addition applies to any number of components and this was exploited to create a multicomponent analysis. A change in phasor distribution towards free or bound NADH can then be separated and analyzed independent of the third individual. Law of phasor addition also enables calculation of the fraction of the third species and helps in quantifying the differential distribution and accumulation of lipids in different cell/ tissues. This approach has been employed to study lipid deposition in mice liver tissues from mice undergoing high fat diet. We envision the use of multicomponent phasor distribution in different scenarios, including multicomponent mixtures, similar to lipid mixtures under cholesterol treatment/ depletion and binding of dyes to different areas and components of cells. (Work supported by NIH-P41_GM103540 and NIH-P50_GM076516).