FLOW CYTOMETER FOR QUANTIFYING FLUORESCENCE LIFETIME

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The long-term goal of this project is the development of advanced flow cytometric methods for measuring phase-resolved fluorescence emissions and excited-stated lifetimes on fluorochromes bound to cells and chromosomes. The specific aims of this project are 1) to apply the technology to a wide range of biological systems that take advantage of these unique measurement capabilities; 2) determine the limits of the technology for detecting and measuring low-level emission signals from fluorescent probes in backgrounds caused by cellular autofluorescence, by spectral emission overlap among fluorescence detection channels, by unbound/nonspecific fluorophore labeling, and by Raman/Rayleigh scatter; and 3) to improve and advance the technology for making phase-resolved multicolor fluorescence and lifetime measurements using single- or dual-modulated laser excitation. The phase-sensitive flow cytometer project is relatively new so that many of its potential applicati ons have not been fully explored and developed. However, because it can separate fluorescence emissions both electronically and optically, quantify lifetime(s) directly as a parameter, and also make conventional flow cytometric measurements, it has a wide range of technically possible applications. This new technology will increase the range of fluorescent markers that can be used in multi-labeling applications, yield more accurate results by enhancing measurement precision and sensitivity and reducing background interferences, and through biomedical research, the technology will significantly expand the researchers' understanding of biological processes at the cellular, subcellular, and molecular level. A Research Highlight in this report describes the use of fluorescence lifetime as a means of distinguishing between fluorophors when the emission spectra are overlapping.