Phase-Sensitive Flow Cytometry: New Technology For Analyzing Biochemical, Functional, and Structural Features in Fluorochrome-Labeled Cells/Particles

Abstract

Flow cytometry (FCM) instruments rapidly measure biochemical, functional, and cytological properties of individual cells and macromolecular components, e.g., chromosomes, for clinical diagnostic medicine and biomedical and environmental research applications. These measurements are based on labeling cells with multiple fluorochromes for correlated analysis of macromolecules, such as, DNA, RNA, protein, and cell-surface receptors. In addition to utilizing the spectral emission properties of fluorescent markers, i.e., different colors/intensities, to measure specific cellular features, the excited state lifetimes also can provide a means to discriminate among the different fluorochromes. A new FCM approach, based on phase-resolved fluorescence lifetime spectroscopy methods (Vesoelova et al, 1970, Lakowicz and Cherek, 1981), recently has been developed to provide unique capabilities for separating signals from multiple overlapping emissions in fluorochrome-labeled cells as they pass across a modulated laser excitation source (Steinkamp and Crissman, 1993). In addition, the measurement of fluorescence lifetime (Pinsky et al, 1993, Steinkamp et al, 1993) also is of importance because it provides information about fluorophore/cell interactions. An important advantage of lifetime measurements is that lifetimes in some case can be considered as absolute quantities. However, the lifetime of fluorophores bound to cellular macromolecules can be influenced by physical and chemical factors near the binding site, such as solvent polarity, cations, pH, energy transfer, excited-state reactions, and quenching. Thus, lifetime measurements can be used to probe the cellular environment, possibly including chemical and structure changes that occur in DNA and chromatin. Table I lists the lifetimes of some typical fluorochromes that are used to quantify cellular DNA, total protein, and antibody-labeling to cellular antigens.