The evaluation of complex metabolic changes of individual live cells and heterogeneous cell cultures is not feasible using traditional methods due to their destructive behavior and lack of spatial information. Two-photon excited fluorescence of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) facilitate a label-free and non-destructive evaluation of metabolic activity. This study explores the phasor approach in combination with two-photon fluorescence lifetime imaging microscopy (FLIM) as a potential method to evaluate pharmacologically induced metabolic changes that occur during the browning of adipocytes. The possibility of browning of white adipose cells is a desirable prospect for the treatment of obesity and related disorders. Here, we compared the results obtained by Fourier-based phasor analysis with the traditional exponential FLIM analysis as well as results of an extracellular flux analyzer. The alteration of glycolytic function and oxidative phosphorylation after treatment with pharmacological reagents significantly shift the contribution of each of the fluorescence lifetime components to the total fluorescence intensity. Further, we showed that the ratios of the lifetime components obtained by the phasor approach reflect the shift in mitochondrial and cytosolic NADH concentration. The phasor analysis agrees with traditional assessments, such as the optical free-to-bound NADH ratio as well as the oxygen consumption rate and extracellular acidification rate as determined by the extracellular flux analyzer. Our results support the concept that non-invasive sensing of fat metabolism and browning of fat may be possible by analyzing the fluorescence lifetime of NADH using the phasor approach.
Michael Evers, Nunciada Salma, Sam Osseiran, Malte J. Casper, Reginald Birngruber, Conor L. Evans, and Dieter Manstein, "Phasor fluorescence lifetime microscopy of NADH to analyze metabolic activity of adipocytes (Conference Presentation)," Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 1049808 (Presented at SPIE BiOS: January 28, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2289294.5751630831001.
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