|
We present a cytometric and metabolic screening tool that measures shifts in NAD(P)H autofluorescence and autofluorescence lifetimes from single cells based on metabolite-enzyme interactions. Short autofluorescence lifetimes of NAD(P)H (~0.1-1ns) indicate the metabolite is unbound from metabolic enzymes and the cell is favoring glycolysis for energy production. In contrast, longer autofluorescence lifetimes of NAD(P)H (~1-7ns) are an inference that the metabolite is bound to metabolic enzymes and the cell is respiring under oxidative phosphorylation. Using a simple time-resolved flow cytometer we are able to measure autofluorescence lifetimes of MCF-7 and T47D breast cancer cells, which we relate to metabolic changes within each cell line. In order to determine the resolution limits of our time-resolved instrument, we first treated cells under different conditions that directly alter the metabolic pathway that drives their energy production. We deprived cells of serum in their growth media, which drives the cell to utilize glycolysis as a metabolic pathway. By comparing normal to deprived cells, we were able to determine if our cytometry system is able to measure differences in the autofluorescence lifetimes. Results show a decrease in lifetime and autofluorescence intensity for both T47D and MCF7 following serum deprivation. Initial cytometric analysis illustrates consistent lifetime data with respect to fluorescence lifetimes and fluorescence intensities decreasing as expected. This study is a preliminary confirmation that our timeresolved cytometer can effectively detect autofluorescence signals, albeit with some limitations in lifetime resolution. Future work will include refinement of lifetime analysis, frequency domain approaches, and improving sensitivity.
|
