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

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 pnnciples to provide unique capabilities for making phaseresolved 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 photomultipher tube detector The modulated detector output signals, which are shifted in phase from a reference signal and amplitude demodulated, are processed by phase-sensitive detection electronics to resolve signals from heterogeneous emissions and quantify decay lifetimes directly The output signals are displayed as frequency distnbution histograms and bivanate diagrams using a computer-based data acquisition system 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 I to 30 MHz, 3) fluorescence measurement precision of 1 3% on alignment fluorospheres and 3 4% on propidium iodide (P1)-stained cells, 4) the resolution of P1 and FITC signals from cells stained in combination with P1 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. 1. INTROPUCTIQN 0-8194-111 2-4/93/$4.00 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/23/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx

Investigation into phase-resolved optical technique for latent fingerprint detection and bioimaging

“…The modulation factor, , phase sift m φ and angular modulation frequency are related through following equations as [1,[52][53][54][55],…”