Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase‐sensitive flow cytometry

Abstract

A flow cytometric method has been developed that uses phase-sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a longpass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual-channel phase-sensitive detection electronics and the phase-resolved PIand FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by m/2 + degrees and the phase of the other detector channel reference signal by -m/ 2 + +z degrees, where and +z are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase-sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering. Key terms: Fluorescence lifetime, phasesensitive detection, propidium iodide, fluorescein isothiocyanate, fluorescence phase-resolved measurement Flow cytometry is a n important clinical diagnostic tool for use in immunology, hematology, and oncology, including its application to basic biomedical research. Clinical tests and biological experiments often require the labeling of cells with multiple fluorochromes for correlated analysis of cellular properties. A major limitation of these procedures is the availability of fluorescent dyes with a common excitation region, i.e., requiring only one excitation source, and emission spectra that are sufficiently separated to permit measurement by multicolor detection methods (18) that employ dichroic and bandpass filters. To alleviate the problem, multiple excitation sources are usually employed (17) to excite sequentially cells labeled with fluorochromes that have separated excitation spectra. This approach has greatly increased the number of fluorochromes available for multicolor labeling studies, but the instrumentation has become increasingly complex in both design and function.We have developed a new flow cytometric method (19) to separate signals from fluorochromes by phaseresolved measurement of fluorescence emission signals. Our intention is to resolve signals from fluorochromes with overlapping emission spectra, but 'Research reported in this article was performed under the auspices of the U.S. Department of Energy.

Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

“…This effect leads to potential preselection of specific lifetime value for a probe through a combination of dye concentration and solvent, either PBS or D 2 O-saline. The use of multiple DNA fluorochromes has been used previously to elucidate changes throughout the cell cycle (Steinkamp and Crissman 1993) and for the determination of apoptotic populations (Darzynkiewicz et al 1992). Because many of these fluorochromes have overlapping emission spectra, their use in combination for conventional flow cytometry is limited.…”

“…Because many of these fluorochromes have overlapping emission spectra, their use in combination for conventional flow cytometry is limited. However, because dyes with overlapping emission spectra, such as PI, EB, and EthD II, have different lifetime values, the potential exists for use of these dyes in combination for phase-resolved measurements of fluorescence (Steinkamp and Crissman 1993). Information on the dye-dye self-quenching of the various fluorochromes will allow the determination of optimal dye concentrations to use for studies on the dyes in combination.…”

“…Fluorescence lifetime measurements were performed on the Los Alamos phase-sensitive flow cytometer which has been described in detail (Steinkamp and Crissman 1993;. Stained cells were excited by a cw laser beam from an Argon ion laser (Model 2025; Spectra Physics, Palo Alto, CA), with the intensity being sinusoidally modulated at 29 MHz.…”

“…Measurement of fluorescence lifetime using phasesensitive electronics has recently provided an additional parameter for flow cytometry analysis (Deka et al 1994;Pinsky et al 1993;Steinkamp and Crissman 1993;). Phase-sensitive flow cytometry provides the capability to quantify fluorescence lifetimes and to resolve the emissions of multiple fluorochrome labels that have different lifetime values (Steinkamp and Crissman 1993).…”

“…Phase-sensitive flow cytometry provides the capability to quantify fluorescence lifetimes and to resolve the emissions of multiple fluorochrome labels that have different lifetime values (Steinkamp and Crissman 1993). Quantitation of the lifetime of DNAbound fluorochromes provides information on the molecular interactions of a fluorochrome within the target molecule (Sailer et al 1996;Heller and Greenstock 1994).…”