Yoko Miura
University of Luebeck
Germany
Title: Monitoring of high glucose-induced cell metabolic alteration of retinal microvascular endothelial cells with fluorescence lifetime imaging microscopy
Biography
Biography: Yoko Miura
Abstract
Retinal microvascular endothelial (RME) cells play a significant role in retinal function as the inner blood-retinal barrier (BRB) whose disruptionis largely relevant to theearly pathological alterations of diabetic retinopathy (DR). Therefore, it is of great importance to elucidate the mechanisms of metabolic and functional alterations of RME cells under diabetic condition, namely under high glucose condition. Although many in vitro and in vivo basic studies have been performed and reported based on this motivation to date, it is still a challenging to monitor the cell metabolisms and functions with living cells. Fluorescence lifetime imaging microscopy (FLIM) is a technique to measure and map the fluorescence lifetime of the fluorophores. Fluorescence lifetime is a fluorophore-intrinsic and moreover, may be influenced by the molecular environments such as temperature, pH, viscosity and molecular protein binding status. FLIM coupled with two-photon microscopy (TPM) enables to measure the fluorescence intensities as well as fluorescence lifetimes of the auto fluorescence of two different coenzymes that are important in cell metabolisms, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH). With TPM-FLIM, the redox ratio of NADH/FAD as well as fluorescence lifetimes of NADH (free/protein bound) and FAD (free/protein bound) can be measured. Since these values are largely related to the status of cell metabolisms, it can be used as a non-invasive monitoring method of cell metabolisms. In this study, I will introduce our in vitro experimental results with cultured human RME cells showing the altered intracellular status of NADH and FAD detected with TPM-FLIM under different high glucose conditions. Combined with the results of other experimental procedures to detect cellular oxidative stress or stress-induced protein expressions, TPM-FLIM was found to be a very sensitive and non-invasive method to monitor the status of these metabolisms-related coenzymes in the cells under high glucose conditions. This method might expand the possibility of detecting cell metabolic states in diabetes basic research and eventually in clinical diagnosis.