UV-excited autofluorescence spectroscopy can provide information on the metabolic status of cellular systems, but applications to turbid media such as tissues can be complicated by the presence of multiple scattering, intrinsic absorption, and background fluorescence. Our broader aim is the sensing of cellular-level metabolic status in tissue based the real time assessment of autofluorescence signals using spectral phasor analysis. Previously, we analyzed metabolic responses in yeast cells embedded in turbid media containing significant background fluorescence from collagen. Not only were changes in metabolism detectable under these conditions, but responses associated with NADH- and NADPH-linked metabolisms could also be distinguished. NADH and NADPH are metabolic co-factors having nearly identical excited-state emission but playing significant and distinct roles in cellular metabolism. Here, we extend the phasor analysis approach by sensing metabolic responses of yeast cells embedded in turbid media containing hemoglobin as a source of optical absorption. A metabolic response is induced by chemical perturbation, e.g., by adding cyanide to inhibit cellular respiration or by adding peroxide to induce oxidative stress. We demonstrate that phasor analysis is a versatile tool, e.g., by showing that spectral response associated with changes to cellular metabolism versus optical absorption are spectrally distinct and cannot be accounted for using a two-component spectral model.
The reduced pyridine-nucleotides nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are ubiquitous metabolic cofactors playing significant, distinct roles in cellular metabolism. NADH and NADPH are primarily involved in cellular respiration and in maintaining antioxidant defenses, respectively, however their nearly identical fluorescence properties (the abbreviation NAD(P)H denotes this uncertainty) pose a challenge when interpreting and distinguishing autofluorescence signals. For sensing in turbid media such as tissue, additional challenges include the presence of multiple scattering, intrinsic absorption, and background fluorescence. Here, we assess an approach for distinguishing cellular-respiration and oxidative-stress responses when sensed in turbid media. Spectral phasor analysis, an analytical approach originally developed for the rapid segmentation of hyperspectral images, has been used on UV-excited autofluorescence for the real time monitoring of cellular NAD(P)H conformation. We showed previously that the spectral response to chemicals affecting NADH and NADPH pathways, e.g., in response to cyanide and hydrogen peroxide, does not follow two-component behavior and so could be distinguished in cell-only preparations. Here, we demonstrate pathway-level sensing in turbid media by monitoring the metabolic response of yeast cells embedded in a source for background emission. The distinguishability of UV-excited autofluorescence spectra to chemical perturbations affecting cellular respiration and oxidative stress are compared with previously reported cell-only observations.
Autofluorescence spectroscopy can provide information on the metabolic status of cellular systems, but extensions of these techniques to turbid media such as tissues is complicated by the presence of multiple scattering, background fluorescence, and intrinsic absorption. Phasor analysis is a class of analytical approaches for the real-time assessment of emission signals that could be used to decipher cellular-level metabolic status of tissues. Spectral phasor analysis was originally developed for the rapid segmentation of hyperspectral images and has since been used for monitoring cellular NAD(P)H conformation from UV-excited cellular autofluorescence. Specifically, we showed previously that chemically induced autofluorescence responses in Saccharomyces cerevisiae (baker’s yeast) suspensions could not be accounted for using the two-component free vs. protein-bound model for conformation. Rather, by considering a series of physically similar and dissimilar chemicals acting on multiple metabolic pathways, we showed that responses affecting different pathways, e.g., involving cellular respiration versus oxidative stress, could be distinguished. Here, we seek to extend this pathway-level interpretation to the sensing of cellular metabolism in tissues by monitoring the cyanide-induced metabolic response of yeast cells embedded in media containing 9-cyanoanthracene or collagen as sources of background emission. Despite the similarity between autofluorescence and background spectra, we observe spectral behavior consistent with the discrimination of the metabolic response from the background emission. Performance over specifically selected noncontinuous spectral bands to reject chromophore absorption is also assessed.
Tissue fluorescence spectroscopy and imaging are being investigated as potential methods for non-invasive detection of pre-neoplastic change in the lung and other organ systems. A substantial contribution to tissue fluorescence is known to arise from endogenous cellular fluorophores. Using steady-state and time-resolved fluorescence spectroscopy and imaging, we characterized the endogenous fluorescence properties of immortalized and carcinogen-transformed human bronchial epithelial cells. Non-invasive sensing of endogenous molecular biomarkers associated with human bronchial pre-neoplasia will be discussed.
Fluorescence lifetime imaging microscopy (FLIM) using near-UV excitation is being developed to probe endogenous fluorophores in cells and tissues. Here, we describe such a UV-FLIM system and present a useful method for measuring the fluorescence lifetime discrimination of the system using the viscosity dependent lifetime of 1,4-Bis(5-phenyloxazol-2-yl)benzene (POPOP). The time-domain UV-FLIM system employed a nitrogen laser (337.1 nm) fiber-optic coupled to an inverted microscope. Wide-field fluorescence images were obtained at controlled time delays with a 200 ps gated, intensified-CCD camera. Lifetimes were calculated from the intensity decay on a pixel-by-pixel basis. The system was capable of imaging endogenous fluorescence in living cells using UV excitation. POPOP is a nanosecond lifetime standard suitable for UV excitation (325-375 nm). Its single-exponential lifetime (1.4 ns in ethanol) is comparable to endogenous lifetime values measured in living cells. Increasing solvent viscosity via the incremental addition of glycerol produced a series of POPOP lifetime standards having a range of 1 ns. The FLIM system’s ability to discriminate lifetime differences of 50 ps was demonstrated using the POPOP series. Thus, POPOP’s viscosity dependent lifetime represents a useful and convenient resolution standard for UV-FLIM calibration.
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