The long-term goal of this project is to develop advanced flow cytometric methods to measure phase-resolved florescence emissions and excited-state lifetimes of fluorescent probes bound to macromolecules in cells and chromosomes by phase-sensitive detection. In addition, we propose to apply the technology to biological systems that take advantage of these unique measurement capabilities and thus demonstrate the efficacy of the technology for applications in biomedical research. A first-generation, phase-sensitive cytometer has been developed which combines flow cytometry and frequency-domain fluorescence lifetime spectroscopy principles to provide unique capabilities for making lifetime-based sensing measurements on cells labeled with fluorescent probes, while maintaining the capability to make conventional flow cytometric measurements.
The specific aims of this proposal are to: 1) advance the data acquisition technology for making excited-state lifetime and phase- resolved fluorescence emission measurements by implementing a digital signal processing-based (DSP) system; 2) utilize the phase-sensitive detection technology to reduce or eliminate background interferences in flow cytometric measurements caused by cellular autofluorescence, by unbound (non-specific) fluorophore labeling, by fluorescence spectral emission signal-crosstalk between measurement channels, by light scatter in fluorescence detection channels of multi-laser excitation measurements, and by laser-excitation of the acceptor fluorophore in donor/acceptor fluorescence resonance energy transfer measurements; and 3) apply the technology to biological systems that can take advantage of these unique measurement capabilities to demonstrate the efficacy of the technology for application to biomedical research. We propose to take advantage of progress made during the previous grant period and the technical expertise in our laboratory for design/construction of complex digital/analog electronics, cell-cycle analyses, radiation damage and repair biology, chromosomes and chromatin structure, cell-surface receptor architecture, cellular damage mechanisms (e.g., oxygen free radicals), and DNA, RNA, and protein cytochemistry to test the efficacy of the technology for application to biological and biomedical research problems that will contribute to improving diagnoses, treatment, and to understanding the mechanisms of human diseases.
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