Two-Photon Fluorescence Lifetime Imaging of Natural Coenzymes in Living Cells as a Function of Oxidative Stress

Abstract

Mitochondria play vital roles in energy metabolism, apoptosis, oxidative stress, aging, and neurodegenerative disease [1]. In this contribution, we probe different aspects of cellular response to chemical-induced oxidative stress in living C3H10T1/2 cells using hydrogen peroxide, rotenone, and excess glucose. Using two-photon fluorescence lifetime imaging microscopy (2P-FLIM), we exploit the autofluorescence dynamics of natural coenzymes such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and flavoproteins as intrinsic biomarkers for oxidative stress. The effects of polarization selectivity in 2P-FLIM measurements are being investigated towards the development of a quantitative, genuine non-invasive 2P-FLIM of patho-physiological changes in living cells. The efficiency of 2P-FLIM cellular autofluorescence for monitoring changes in the metabolic and redox states of the cells is compared with conventional assays such as MitoSOX Red, JC-1, and Rhodamine-123 that are routinely used for oxidative stress studies. Our results help in the collective effort to establish cellular autofluorescence as a natural biomarker for biological and biomedical studies.1. Heikal, A.A. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. Biomarkers in Medicine, 4(2): 241-63 (2010).