Using in vivo multiphoton fluorescence lifetime imaging to unravel disease-specific changes in the liver redox state

Abstract

Multiphoton fluorescence lifetime microscopy has revolutionized studies of pathophysiological and xenobiotic dynamics, enabling the spatial and temporal quantification of these processes in intact organs in vivo. We have previously used multiphoton fluorescence lifetime microscopy to characterise the morphology and amplitude weighted mean fluorescence lifetime of the endogenous fluorescent metabolic cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) of mouse livers in vivo following induction of various disease states. Here, we extend the characterisation of liver disease models by using nonlinear regression to estimate the unbound, bound fluorescence lifetimes for NAD(P)H, flavin adenine dinucleotide (FAD), along with metabolic ratios and examine the impact of using multiple segmentation methods. We found that NAD(P)H amplitude ratio, and fluorescence lifetime redox ratio can be used as discriminators of diseased liver from normal liver. The redox ratio provided a sensitive measure of the changes in hepatic fibrosis and biliary fibrosis. Hepatocellular carcinoma was associated with an increase in spatial heterogeneity and redox ratio coupled with a decrease in mean fluorescence lifetime. We conclude that multiphoton fluorescence lifetime microscopy parameters and metabolic ratios provided insights into the in vivo redox state of diseased compared to normal liver that were not apparent from a global, mean fluorescence lifetime measurement alone.

Synthesis, Characterization, and Photophysical Properties of a New Indolizine Pyridine Complex with a Large Two‐Photon Absorption Cross‐Section

“…Fluorescence lifetime measurement plays an important role in the application of non-radiative energy transfer and timeresolved fluorescence anisotropy. [44,45] At the same time, fluorescence lifetime measurement has an important influence on the understanding of energy level structure and the excited-state relaxation time of materials. [46] We tested the fluorescence lifetime of fluorescent compound 1.…”

Fatty Acids and Bilirubin as Intrinsic Autofluorescence Serum Biomarkers of Drug Action in a Rat Model of Liver Ischemia and Reperfusion

“…The red emission of porphyrins, in turn, can reflect an altered metabolism of heme [11][12][13], while NAD(P)H and flavins, respectively, fluorescing in the reduced and oxidized state, have been the mostly considered AF biomarkers of the redox state of cells for a long time. The strict engagement of these coenzymes of energy metabolism, reductive biosynthesis, and antioxidant activities has made their AF a candidate for both in-vivo monitoring of liver functionality under normal or physiologically altered conditions, or in-situ disease detection [4,[14][15][16][17][18][19][20][21][22]. Only some free fatty acids are capable of fluorescing under the near-UV-blue light excitation.…”

Special issue on fluorescence lifetime imaging (FLIM): from fundamentals to applications

“…Barkauskas et al study mouse livers in vivo following induction of various disease states and show that NAD(P)H amplitude ratio, and the FAD/NAD(P)H fluorescence lifetime redox ratio can be used as discriminators of diseased liver from normal liver. The redox ratio provided a sensitive measure of the changes in hepatic fibrosis, biliary fibrosis and hepatocellular carcinoma [4].…”