Flow cytometry is a powerful cell analysis tool, providing information on cell count, shape, size, DNA content, redox state, membrane permeability, and surface receptors, among other features. It can be used on both live and fixed cells; and can be used to non-destructively sort cells based on these characteristics. Flow cytometry has utility in research and clinical fields as diverse and important as tumor biology research, cancer and AIDS diagnostics, stem cell therapy, immunophenotyping, and cancer immunotherapy. Flow cytometry relies extensively on fluorescent labeling cells; with the emission light collected from the interrogation region divided spectrally by filters and beam splitters, and directed to detectors that measure intensity. One common problem derives from the fact that the exogenously added fluorophores are not the only source of fluorescence in the sample. So-called cellular autofluorescence can be, in some circumstances, a major contributor to the signal present in each of the detection channels. A number of solutions have been proposed, but they often involve complex experimental protocols, or complex processing of data. This leaves the field wanting for a solution to the nagging problem of interference from cellular autofluorescence. Here we propose to use fluorescence lifetime as a second discriminating parameter in order to distinguish autofluorescence interference from the desired signal. We propose to build a simple system using a single pulsed laser, which will be sufficient to demonstrate feasibility of our method. The system will include a high- speed pulsed 375-nm laser capable of 0.5-ns pulses and the required fast electronics and 3 emission channels, which span the range of the broad cellular autofluorescence. We will leverage the deep expertise of Kinetic River in designing and building complex, innovative cytometry instruments; implementing novel hardware solutions to enable lifetime measurements in flow cytometry; and innovative, proprietary, and patent-protected techniques for rapid analysis of complex lifetime decay curves. We will be assisted by consultants and collaborators with deep experience in fluorescence lifetime measurement, autofluorescence signals, cell-based assays and algorithm development. To demonstrate the feasibility of our approach, we are pursuing the following strategy: (1) build prototype instrument and optimize system hardware; (2) characterize instrument performance; and (3) validate using relevant biological samples. Successful completion of this work will pave the way for further development in Phase II, which will see the addition of other laser lines with lifetime capabilities to create a general purpose instrument. This instrument will be similar in cost to similarly equipped cytometers, but will have the advantage of automated elimination of cellular autofluorescence. Such an instrument will have wide-ranging utility, increased ease of use, and will allow for clearer interpretation of experimental results.
Cellular autofluorescence is ever-present in both live and fixed cells. In flow cytometry, this can interfere with the desired signal and cause loss of sensitivity or erroneous results. We are developing a method, based on fluorescence lifetime, that can automatically eliminate autofluorescence from standard flow cytometry assays.
