Collaborative Research: EAGER: Autofluorescence lifetime flow sorting with time-correlated single photon counting

The goal of this project is to develop technologies that can identify and separate cell type based on their internal chemistry. Cells exhibit different amounts of fluorescence based on the presence of certain molecules. Knowledge of these molecules can be used to confirm cell properties such as metabolic activity. This knowledge can be used to determine that stem cells are healthy and viable for transplantation for example. In this project the approach will be used to study and identify adult brain stem cells, a cell population that is under study for research into cognitive diseases and may be used as a therapeutic strategy for such diseases. This project will create a new cell sorting technique based on measuring thefluorescence from these molecules to enable improvements in isolating stem cells. Significantly, the technique is label-free and rapid, suggesting that it can be adapated in the work flow for stem cell purification. These concepts are shared with K-12 students through high school apprentices in the lab and an immersive rural summer science camp. Preliminary data from multiphoton fluorescence lifetime imaging studies show changes in the fluorescence lifetime of NAD(P)H, a molecular indicator of cell metabolic activity. New NAD(P)H lifetime flow cytometry technologies have also been developed that can accurately profile the same biological changes captured with standard multiphoton approaches. However, real-time cell sorting capabilities based on NAD(P)H lifetimes are not yet available. Therefore, this proposal will develop new sorting capabilities for NAD(P)H lifetime flow cytometry to (1) create a single cell deposition module that provides cell-to-cell correspondence between NAD(P)H lifetimes and self-renewal capacity to enable label-free identification of stem cells within a mixed population, and (2) separate metabolic subpopulations within stem cell lines for improved quality control of these lines. The development of active sorting technologies based on label-free NAD(P)H fluorescence lifetimes, including binary sorting into bins and single cell deposition systems, would provide a radically different approach for cell enrichment and single cell functional assessments. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.