Phase-sensitive flow cytometer
- Los Alamos National Lab., NM (United States)
A phase-sensitive flow cytometer has been developed that combines flow cytometry and fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved measurements on single cells. Stained cells are analyzed as they intersect an intensity-modulated (sinusoid) laser beam. Fluorescence is measured using only a collecting lens, a longpass filter, and a photomultiplier tube detector. Signals are processed by phase-sensitive detection electronics to resolve signals from heterogeneous emissions and quantify decay lifetimes. Results have demonstrated: (1) signal phase shift and amplitude demodulation on fluorospheres and PI-stained cells; (2) a detection threshold of 800-300 fluorescein molecules equivalence for excitation frequencies 10 to 30 MHz; (3) measurement precision of 1.5% on fluorospheres and 4.0% on Pi-stained cells; (4) the resolution of Pi and FITC signals based on differences in their lifetimes; and (5) the measurement of single decay lifetimes by the two-phase ratio method. The significance of this new technology is that the number of fluorochromes usable in multilabeling experiments will be increased; background interferences (autofluorescence, unbound dye, nonspecific staining, Raman scatter) will be eliminated; and fluorescence lifetime can be quantified to study the interaction of fluorochrome binding.
- OSTI ID:
- 6831810
- Report Number(s):
- CONF-9303114-; CODEN: CYTODQ
- Journal Information:
- Cytometry (Baltimore); (United States), Vol. 6; Conference: 16. congress of the International Society for Analytical Cytology, Colorado Springs, CO (United States), 21-26 Mar 1993; ISSN 0196-4763
- Country of Publication:
- United States
- Language:
- English
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Resolution of heterogeneous fluorescence emission signals and decay lifetime measurement on fluorochrome-labeled cells by phase-sensitive FCM
Resolution of heterogeneous fluorescence emission signals and decay lifetime measurement on fluorochrome-labeled cells by phase-sensitive FCM