onitoring changes of cellular metabolism and icroviscosity in vitro based on time-resolved ndogenous fluorescence and its anisotropy decay ynamics

ei Zheng ong Li ianan Y. Qu ong Kong University of Science and Technology epartment of Electronic and Computer Engineering lear Water Bay, Kowloon ong Kong, China Abstract. Reduced nicotinamide adenine dinucleotide NADH is a well-known metabolic coenzyme and endogenous fluorophore. In this study, we develop a system that simultaneously measures timeand wavelength-resolved fluorescence to extract free and proteinbound NADH signals from total cellular fluorescence. We analyze temporal characteristics of NADH fluorescence in a mixture of NADH and lactate dehydrogenase LDH as well as in living cell samples. The results show that in both the NADH/LDH mixture and cell samples, a fraction of free NADH and protein-bound components can be identified. The extracted free and bound NADH signals are confirmed by time-resolved measurement of anisotropy decay of NADH fluorescence, based on the fact that free NADH is a small fluorescent molecule with much shorter rotational diffusion time than bound NADH. The ratio of free NADH signal to bound NADH signal is very different between normal and cancer cervical epithelial cells. In addition, the ratio changes significantly when the cell samples are treated with a mitochondrial inhibitor or uncoupler, demonstrating that the method is sensitive to monitor cellular metabolic activity. Finally, we demonstrate that the microviscosity for relatively small molecules such as NADH in cells could be extracted from wavelengthand time-resolved NADH fluorescence of living cell samples. © 2010 Society of Photo-Optical Instrumentation Engineers. DOI: 10.1117/1.3449577