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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Research Project (R01)
Project #
2R01RR007855-04
Application #
2283622
Study Section
Special Emphasis Panel (ZRG7-SSS-3 (14))
Project Start
1992-09-30
Project End
1998-09-29
Budget Start
1995-09-30
Budget End
1996-09-29
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Los Alamos National Lab
Department
Type
Schools of Arts and Sciences
DUNS #
City
Los Alamos
State
NM
Country
United States
Zip Code
87545
Cui, H Helen; Valdez, Joseph G; Steinkamp, John A et al. (2003) Fluorescence lifetime-based discrimination and quantification of cellular DNA and RNA with phase-sensitive flow cytometry. Cytometry A 52:46-55
Crissman, H A; Steinkamp, J A (2001) Flow cytometric fluorescence lifetime measurements. Methods Cell Biol 63:131-48
Steinkamp, J A (2001) Time-resolved fluorescence measurements. Curr Protoc Cytom Chapter 1:Unit 1.15
Steinkamp, J A; Valdez, Y E; Lehnert, B E (2000) Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres. Cytometry 39:45-55
Keij, J F; Bell-Prince, C; Steinkamp, J A (2000) Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs. Cytometry 39:203-10
Keij, J F; Bell-Prince, C; Steinkamp, J A (1999) Simultaneous analysis of relative DNA and glutathione content in viable cells by phase-resolved flow cytometry. Cytometry 35:48-54
Steinkamp, J A; Lehnert, B E; Lehnert, N M (1999) Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry. J Immunol Methods 226:59-70
Steinkamp, J A; Lehnert, N M; Keij, J F et al. (1999) Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry. Cytometry 37:275-83
Sailer, B L; Valdez, J G; Steinkamp, J A et al. (1998) Apoptosis induced with different cycle-perturbing agents produces differential changes in the fluorescence lifetime of DNA-bound ethidium bromide. Cytometry 31:208-16
Cobo, J M; Garcia-Canero, R; Valdez, J G et al. (1998) Attenuation of apoptotic DNA fragmentation by amiloride. J Cell Physiol 175:59-67

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