Resolution of heterogeneous fluorescence emission signals and decay-lifetime measurement of fluorochrome-labeled cells by phase-sensitive FCM

John A. Steinkamp; Harry A. Crissman

doi:10.1117/12.144718

18 May 1993 Resolution of heterogeneous fluorescence emission signals and decay-lifetime measurement of fluorochrome-labeled cells by phase-sensitive FCM

John A. Steinkamp, Harry A. Crissman

Author Affiliations +

Proceedings Volume 1885, Advances in Fluorescence Sensing Technology; (1993) https://doi.org/10.1117/12.144718
Event: OE/LASE'93: Optics, Electro-Optics, and Laser Applications in Scienceand Engineering, 1993, Los Angeles, CA, United States

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.

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John A. Steinkamp and Harry A. Crissman "Resolution of heterogeneous fluorescence emission signals and decay-lifetime measurement of fluorochrome-labeled cells by phase-sensitive FCM", Proc. SPIE 1885, Advances in Fluorescence Sensing Technology, (18 May 1993); https://doi.org/10.1117/12.144718

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