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Proceedings Paper

Resolution of heterogeneous fluorescence emission signals and decay-lifetime measurement of fluorochrome-labeled cells by phase-sensitive FCM
Author(s): John A. Steinkamp; Harry A. Crissman
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Paper Abstract

A phase-sensitive flow cytometer has been developed to resolve signals from heterogeneous fluorescence emission spectra and quantify fluorescence decay times on cells labeled with fluorescent dyes. This instrument combines flow cytometry (FCM) and fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved measurements on single cells in flow, while preserving conventional FCM measurement capabilities. Stained cells are analyzed as they pass through an intensity-modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using a collecting lens, a longpass barrier filter to block scattered laser excitation light, and a photomultiplier tube detector. Results have demonstrated: (1) signal phase shift, amplitude demodulation, and average measurement of fluorescence lifetimes on stained cells; (2) a detection limit threshold of 300 to 500 fluorescein isothiocyanate (FITC) molecules equivalence for excitation frequencies 1 to 30 MHz; (3) fluorescence measurement precision of 1.3% on alignment fluorospheres and 3.4% on propidium iodide (PI)-stained cells; (4) the resolution of PI and FITC signals from cells stained in combination with PI and FITC, based on differences in their decay lifetimes; and (5) the ability to measure single decay times by the two-phase, phase comparator, method.

Paper Details

Date Published: 18 May 1993
PDF: 12 pages
Proc. SPIE 1885, Advances in Fluorescence Sensing Technology, (18 May 1993); doi: 10.1117/12.144718
Show Author Affiliations
John A. Steinkamp, Los Alamos National Lab. (United States)
Harry A. Crissman, Los Alamos National Lab. (United States)

Published in SPIE Proceedings Vol. 1885:
Advances in Fluorescence Sensing Technology
Joseph R. Lakowicz; Richard B. Thompson, Editor(s)

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