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Regulatory dynamics of energy metabolism in living cells entails a coordinated response of multiple enzyme networks that operate under non-equilibrium conditions. Here we show that mitochondrial dysfunctions associated with the aging process significantly modify nonlinear dynamical signatures in free radical generation/removal, thereby altering energy metabolism in liver cells. We support our data with a plausible biochemical mechanism for modified bioenergetics that involves uncoupling protein-2 that is up-regulated in aged cells as an adaptive response to mitigate increased oxidative stress. Combining high spatial and temporal resolution imaging and bio-energetic measurements, our work provides experimental support to the hypothesis that mitochondria manifest nonlinear dynamical behavior for efficiently regulating energy metabolism in intact cells, and any partial or complete reduction in this behavior would contribute to organ dysfunctions including the aging process and other disease processes.

Background: Spectral Imaging Microscopy is gaining attention in biological research. Most of the commercial systems in vogue employ linear spectral un-mixing algorithms and/or spectral profile matching algorithms to extract the component spectral information from the measured specimen spectra. The need to accurately deconvolve multiple spectra with minimal cross-contamination is always accompanied by an increase in system complexity and cost., Methods: We describe here a variant of the spectral waveform cross-correlation analysis (SWCCA) method where the master reference spectral library is constructed by composite spectra with varying ratios of component spectra, unlike the conventional spectral library where pure spectra form the components. We demonstrate that this spectral kinetics ratiometric approach gives realistic estimates of fluorophore distribution in living cells with a better spectral correlation as compared with pure component spectral libraries., Results: Biological applications demonstrated in this article include acceptor photobleaching FRET, caspase activity during cell death and mitochondrial membrane polarization kinetics during substrate metabolism., Conclusions: Beyond the representative applications presented in this article, we think the proposed approach can be valuable in dynamic studies of a variety of other cellular processes such as pH oscillations, photobleaching and quenching kinetics. Besides giving better spectral correlation and real-time monitoring of biophysical processes in living cells, this method can serve as an economical solution for high-throughput spectral classification requirements., ((c) 2006 International Society for Analytical Cytology.)

Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a subfraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria. [Copyright &y& Elsevier]