Fluctuation analysis of mitochondrial NADH fluorescence signals in confocal and two-photon microscopy images of living cardiac myocytes.

A fluctuation analysis was performed on the reduced nicotine adenine dinucleotide (NADH) fluorescence signal from resting rabbit myocytes using confocal and two-photon microscopy. The purpose of this study was to establish whether any co-ordinated biochemical processes, such as binding, metabolism and inner mitochondrial membrane potential, were contributing to NADH signal fluctuations above background instrument noise. After a basic characterization of the instrument noise, time series of cellular NADH fluorescence images were collected and compared with an internal standard composed of NADH in the bathing medium. The coefficient of variation as a function of mean signal amplitude of cellular NADH fluorescence and bathing media NADH was identical even as a function of temperature. These data suggest that the fluctuations in cellular NADH fluorescence in resting myocytes are dominated by sampling noise of these instruments and not significantly modified by biological processes. Further analysis revealed no significant spatial correlations within the cell, and Fourier analysis revealed no coherent frequency information. These data suggest that the impact of biochemical processes, which might affect cellular NADH fluorescence emission, are either too small in magnitude, occurring in the wrong temporal scale or too highly spatially localized for detection using these standard optical microscopy approaches.