Examining cellular metabolism using two-photon and confocal microscopy of NAD(P)H and flavin autofluorescence

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Cells metabolize nutrients to generate and store energy for life. Critically, cell metabolism is dynamic and adapts to nutrient availability in order to survive. Metabolism has classically been studied by grinding-up cells to measure biochemical composition in a test-tube. However, this approach requires large numbers of cells and cannot be collected repetitively on the same sample since it is a terminal event. Our research program takes advantage of an alternative approach that uses an inherent property of every living cell: a change in metabolism modifies the inherent fluorescent properties of a cell (autofluorescence). We use this link between autofluorescence and metabolism to understand how cells respond and adapt to various treatments. To measure autofluorescence from cells in a mouse tissue, we use a microscope with a variety of different coloured lasers. Importantly, we can repetitively measure the autofluorescence from a single sample, which allows us to measure the response to a specific treatment over a period of time. In the first proposed study, we use autofluorescence microscopy to determine how cell metabolism changes in response to the deprivation of oxygen. In the second study, we use other available optical sensors to compare with autofluorescence and further examine metabolism. The third study develops a novel sensor to gather specific comparative data on multiple processes of metabolism. These studies will increase our understanding of how cellular metabolism changes in response to the surroundings and lead to new tools to simultaneously and dynamically measure various aspects of metabolism.