(622af) Using the Fluorescence Lifetime As a Cytometric Parameter to Sort Unlabeled, Heterogeneous Populations of Cells

Time-resolved flow cytometers enable the measurement of the fluorescence lifetime as a singular parameter to count and sort cells.  The fluorescence lifetime is the average time a fluorescent molecule spends in its excited state prior to relaxation to the ground state; it is a unique photophysical trait that, with time-resolved detection systems, can provide quantitative information for cellular assays.  In the case of biomedical assays using proteins, polymers, fluorophores, chromophores, quantum dots, nanoparticles, and other targets for diagnostics, instruments that provide sensitive relaxation kinetic measurements may help bridge qualitative with quantitative information.  Yet it is well known that to observe an excited state is difficult; radiative decay happens over nearly infinitesimal (e.g., nanoseconds to near-instantaneous) timescales.  Moreover, it is non-trivial to capture heterogeneous time-resolved information from several different excitable molecules when excitation is observed from single cells and particles in fluidic states.   Work by our laboratory has focused on making high throughput multi-exponential fluorescence decay detection possible in flow cytometry.  In this contribution we demonstrate the utility of fluorescence lifetime measurements for cell sorting in a label-free fashion.  That is, cells are detected based on their autofluorescence expression.  The fluorescence lifetime of the autofluorescence specie, NADH, is measured and used as a sorting parameter.  A range of mammalian cells were taken for this demonstration, including normal breast cells, breast cancer cells, and lymphocytes.  We mix various populations of these cells to prove discrimination based on the NADH lifetime, which shifts distinctly when in a bound state during metabolic alteration (highly proliferative cell states vs. quiescent). The NADH lifetime shifts measured range from 1.1 to 2.4 nanoseconds, and the sorting purity ranged depending on the relative mixtures of the tumorigenic and non-tumorigenic cells.  We aim to use label-free, lifetime sorting as a way to screen for circulating tumor cells, rapidly and without the need to pre-treat extracted blood cells from the body during this diagnostic procedure.