The long-term goal of this project is to develop advanced flow cytometric methods for measuring phase-resolved fluorescence emissions and excited- state lifetimes on fluorochromes bound to macromolecular components in cells and chromosomes by phase-sensitive detection. A first-generation, phase-sensitive flow cytometer has been developed that combines flow cytometry and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making phase-resolved measurements on fluorochrome-labeled cells and subcellular components (chromosomes). No other instrument can resolve and measure fluorescent probe emissions based on differences in their lifetimes and quantify lifetime directly in real time, while maintaining the capability to made conventional flow cytometric measurements. In the prototype system, cells/chromosomes labeled with fluorescent probes are analyzed as they intersect a high- frequency (sine wave), intensity-modulated laser beam. Modulated fluorescence signals are processed by phase-sensitive detection electronics to resolve signals from overlapping probe emissions and to quantify lifetime as a new parameter.
The specific aims of this proposal are: 1) to apply the technology to a wide range of biological systems take advantage of these unique measurement capabilities; 2) to 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 upon and advance the technology for making phase-resolved multicolor fluorescence and lifetime measurements using single- or dual-modulated laser excitation. 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. We propose to take advantage of progress made during the previous grant period and expertise in our laboratory for cell-cycle analyses, cytogenetics, chromosomes and chromatin structure, cell-surface receptor architecture, pulmonary damage mechanisms (cell-cycle related), and DNA, RNA, and protein cytochemistry to test the efficacy of the technology for future application to diverse biological and biomedical research problems that will contribute to improving diagnoses, treatment, and further the understanding of mechanisms that underlie a variety of human diseases.
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