Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD + reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of proteinbound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD + . Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD + ratio.
Multiphoton fluorescence lifetime imaging microscopy (FLIM) is a noninvasive, cellular resolution, 3-D functional imaging technique. We investigate the potential for in vivo precancer diagnosis with metabolic imaging via multiphoton FLIM of the endogenous metabolic cofactor nicotinamide adenine dinucleotide (NADH). The dimethylbenz[alpha]anthracene (DMBA)-treated hamster cheek pouch model of oral carcinogenesis and MCF10A cell monolayers are imaged using multiphoton FLIM at 780-nm excitation. The cytoplasm of normal hamster cheek pouch epithelial cells has short (0.29+/-0.03 ns) and long lifetime components (2.03+/-0.06 ns), attributed to free and protein-bound NADH, respectively. Low-grade precancers (mild to moderate dysplasia) and high-grade precancers (severe dysplasia and carcinoma in situ) are discriminated from normal tissues by their decreased protein-bound NADH lifetime (p<0.05). Inhibition of cellular glycolysis and oxidative phosphorylation in cell monolayers produces an increase and decrease, respectively, in the protein-bound NADH lifetime (p<0.05). Results indicate that the decrease in protein-bound NADH lifetime with dysplasia is due to a shift from oxidative phosphorylation to glycolysis, consistent with the predictions of neoplastic metabolism. We demonstrate that multiphoton FLIM is a powerful tool for the noninvasive characterization and detection of epithelial precancers in vivo.
When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope. Fluorescence lifetime images from 16 individual wavelength components of the emission spectrum can be acquired with 10-nm resolution on a pixel-by-pixel basis. The instrument provides a unique visualization of cellular structures and processes through spectrally and temporally resolved information and may ultimately find applications in live cell and tissue imaging.
A major obstacle in the race to develop two-photon fluorescence endoscopy is the use of complicated bulk optics to transmit an ultrashort-pulsed laser beam and return the emitted fluorescence signal. We describe an all-fiber two-photon fluorescence microendoscope based on a single-mode optical fiber coupler, a microprism, and a gradient-index rod lens. It is found that the new endoscope exhibits an axial resolution of 3.2 microm and is capable of imaging transverse cross sections of internal cylindrical structures as small as approximately 3.0 mm in diameter. This device demonstrates the potential for developing a real-time diagnostic tool for biomedical research without the need for surgical biopsy and may find applications in photodynamic therapy, microsurgery, and early cancer detection.
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