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29 April 1998Progress toward combined phase and amplitude demodulation fluorescence lifetime measurements by flow cytometry
A phase-sensitive flow cytometer has been developed to quantify fluorescence lifetimes directly as a parameter in real time on particles and cells labeled with fluorophores. This instrument combines flow cytometry and frequency-domain fluorescence lifetime spectroscopy measurement principles to provide unique features for making excited-state lifetime measurements. Cells are analyzed as they intersect a high- frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by analog phase comparator measurement electronics to provide output signals proportional to the sine and cosine of the phase difference between the modulated signal input and a steady state reference signal. The ratios, which are proportional to the lifetimes, are displayed as frequency distributions histograms. In this study we present the underlying theory to measure fluorescence lifetimes by both phase shift and amplitude demodulation. Prototype analog amplitude demodulation electronics have been constructed and are being tested using simulated signals, fluorescent microspheres, and cells labeled with fluorescent probes.
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John A. Steinkamp, Jimmie D. Parson, Jan F. Keij, "Progress toward combined phase and amplitude demodulation fluorescence lifetime measurements by flow cytometry," Proc. SPIE 3260, Optical Investigations of Cells In Vitro and In Vivo, (29 April 1998); https://doi.org/10.1117/12.307098