Use of Time-Resolved Flow Cytometry to Detect Metabolic Shifts in Cancer Cells

Flow cytometry is the process of passing cells individually through the path of a laser beam with the use of a fluidic stream that carries the cell suspension. With laser excitation, molecules that are labeled on the surface or interior of the cell will fluoresce. The fluorescence is detected and in turn used to make interpretations about cellular properties including phenotype, genotype, morphology, and other features. In this research we collect light scatter, fluorescence, and a parameter that is not found on conventional flow cytometers--the fluorescence lifetime. The lifetime is important because it is related to the time it takes for excited molecules to decay, which is affected by the cellular microenvironment. We use a specialty flow cytometer to measure this value (~ 1 – 20 nanoseconds). This research is focused on designing and optimizing the cytometry instrumentation needed for making fluorescence lifetime measurements. To test our system, we culture immortalized cancer cells and measure endogenous fluorescence from the excitation of naturally occurring intracellular molecules. The results we obtain are values of fluorescence lifetimes that shift depending on the cellular metabolism and that change depending on the cell type and metabolic pathway taken by the cancer cell. Our overall goal is to build a tool that will measure fluorescence from the naturally occurring species in the cell (e.g. the NADH metabolite) and look for alterations in the lifetime to identify metabolic shifts in a less invasive manner. At the same time, how the metabolic pathways change once that cancer cells are treated with a drug. And determine which the best treatment to that cancer cell is.