Assessing the Metabolic State of Cultured Cells and the Intact Cochlea by Two-Photon Fluorescence Intensity and Lifetime Imaging

Abstract

The ability to match energy production with demand is a fundamental determinant of the health of cells, tissues, and organs of the body. Metabolism and mitochondrial dysfunction have been broadly implicated in many disease states, including hearing disorders and noise-induced hearing loss. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) and flavoprotein (Fp) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, the fluorescence intensity has commonly been used as an indicator of the reduced NADH and/or oxidized Fp concentration in cells and tissue. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. This presents a new opportunity to track the cellular metabolic state as NADH binding changes with metabolism. To better understand what is revealed by variations in the fluorescence intensity and lifetime distributions, we have used mitochondrial uncouplers and inhibitors in conjunction with manipulation of substrate and oxygen concentrations to systematically adjust the metabolic state. We will compare the similarities and differences of the intensity- and FLIM-based assessment of metabolic rate in model systems of increasing complexity, ranging from single cells to intact tissue, highlighting the advantages and limitations of each technique. Supported by NIH DC 02053, NSF-EPSCoR EPS-0701892 (CFD 47.076), NIH P20 RR16469 from the National Center for Research Resources (NCRR), and NIH R15 GM085776.