Abstract
Every year millions of people in the United States are affected by skin cancer, and like other cancers, the key to an effective treatment is early detection and monitoring of disease progression. To this end, we have used intensity-, time-, and wavelength-resolved multiphoton microscopy to measure changes in cellular metabolism. By analyzing the fluorescence emission of metabolic coenzymes such as the reduced form of Nicotinamide Adenine Dinucleotide (NADH) and the oxidized form of flavin cofactors, slight changes in the mitochondrial microenvironment and cellular metabolism can be quantified. We used dual-channel fluorescence lifetime imaging (FLIM) to quantify metabolic changes both within and between high- and low-HER2 expressing squamous cell carcinoma cell lines (SCC-74B and SCC-74A, respectively). Chemically-induced changes in the metabolic state led to measureable changes in lifetime distributions and fluorescence intensities within each cell line. In addition, SCC-74A and −74B could clearly be distinguished on the basis of the fluorescence intensity dynamic range (difference between uncoupled and inhibited metabolic states) and lifetime distributions. The metabolic imaging technique has also been adapted to living skin in vivo. In particular, fluorescence signals originating from metabolically active and in-active sources are identified and separated based on a combination of the two-photon-excited fluorescence excitation/emission spectra and second harmonic generation (SHG).This study was conducted at the Creighton University Integrative Bio-Imaging Facility (CU-IBIF) and was supported by NIH DC 02053, NSF-EPSCoR EPS-0701892 (CFD 47.076), NIH P20 RR16469 (NCRR) and 5P20GM103427 (NIGMS) as well as the Ferlic Summer Research Program at Creighton University.
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