Fluorescence lifetime measurement of free and cell/particle-bound fluorophore by phase-sensitive flow cytometry

John A. Steinkamp; Jan F. Keij

doi:10.1117/12.307058

1 May 1998 Fluorescence lifetime measurement of free and cell/particle-bound fluorophore by phase-sensitive flow cytometry

John A. Steinkamp, Jan F. Keij

Author Affiliations +

Proceedings Volume 3256, Advances in Optical Biophysics; (1998) https://doi.org/10.1117/12.307058
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

We report new and novel electronics to quantify lifetimes of free (fluorophore solution) and cell/particle-bound fluorophores. This technology combines flow cytometry and frequency-domain fluorescence lifetime spectroscopy measurement principles to provide unique features for making excited-state lifetime measurements of free fluorophore and cell/particle-bound fluorophore on a cell-by-cell basis in real time. Cells labeled with fluorophore and suspended in fluorophore solution are analyzed as they intersect a high- frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence pulse (cells) and steady-state (fluorophore solution) signals are processed by separate phase-sensitive detection channels to quantify lifetimes. The cell-bound fluorophore measurement channel employs a phase comparator to provide two output signals (proportional to the sine and cosine of the phase difference between the signal pulse input and a steady-state reference signal), whereas, the free (solution) fluorophore lifetime measurement channel employs a second phase comparator to provide steady-state sine and cosine outputs which can be gated externally (pulse generator) or by a cell being analyzed. The phase comparator outputs are input to ratio modules for determining the respective lifetimes. Examples of solution and cell/particle lifetime measurements using common fluorophores are described.

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John A. Steinkamp and Jan F. Keij "Fluorescence lifetime measurement of free and cell/particle-bound fluorophore by phase-sensitive flow cytometry", Proc. SPIE 3256, Advances in Optical Biophysics, (1 May 1998); https://doi.org/10.1117/12.307058

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