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Molecular native fluorescence (autofluorescence) can be used as a research tool to understand the underlying mechanisms of molecular interactions and cellular processes under physiological conditions in cells and tissues. Autofluorescent NAD(P)H, and flavins (FAD) are widely utilized as biomarkers for cellular energy metabolism. Tryptophan (Trp) is another endogenous fluorescent biomarker in cancer investigations. Understanding of the intricacies of an array of diseases, from cancer to diabetes to Alzheimer’s disease requires high-resolution imaging techniques such as FLIM, FRET and FLIRR (Fluorescence Lifetime Redox Ratio) for gaining insights to cellular behavior at the molecular level. Fluorescence lifetime imaging (FLIM) is a sensitive technique to investigate NAD(P)H, FAD and Trp in living cancer cells and tissues. The characterization of these endogenous molecules help us to understand the heterogeneous distribution of the metabolic signals or mapping of the metabolic signals in these pathologies. The traditional intensity-based redox ratio includes intensity artefacts, particularly in tissues, due to differential absorption and scattering in tissues and usage of various average excitation intensity levels at different depths. We developed the FLIRR assay, measured by discrete ROIs (1 or 2x2 pixels), mapping cellular morphology and analyzing the heterogeneous environment of the lifetime distribution in prostate cancer cells. Increasing FLIRR levels serve as indicators of changing metabolic states and increased E% between Trp and NAD(P)H was seen at various time durations upon treatment with doxorubicin.
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