Fluorescence Lifetime-Based Discrimination and Quantification of Cellular DNA and RNA with Phase-Sensitive Flow Cytometry

Abstract

Background: Simultaneous measurement of cellular DNA and RNA content provides information for determination of the functional status of cells and, clinically, for the diagnosis and grading assessment of various tumors. Most current flow cytometric methods are based on resolving the fluorescence emission spectra of dyes that bind preferentially to either type of nucleic acid. However, several monochromatic nucleic acid-binding fluorochromes display resolvable differences in fluorescence lifetime when bound to DNA or RNA. The differences in the lifetime of one fluorescent probe provide an alternate means to distinguish the binding of one probe to these cellular macromolecules and to simultaneously measure their cellular contents. Methods: Three nucleic acid intercalating dyes, propidium iodide, ethidium bromide, and ethidium homodimer 1, were selected to study differences in fluorescence lifetimes when bound to cellular DNA and RNA. Fixed HL-60 cells were treated with specific nucleases to initially determine the lifetime values of each dye when bound to the cellular DNA, RNA, or both. The lifetime values were then used as the signatures to resolve the cellular DNA and RNA contents in untreated cells. Results: All three dyes showed fluorescence lifetime differences when bound to RNase-treated, DNase-treated, or untreated cells. With these lifetime values, the fluorescence emissions from DNA, RNA, or DNA/RNA were resolved from untreated cells with the use of phase-sensitive detection. The lifetime differences resulting from the binding to either type of nucleic acid depended on the dye, the staining concentration, and the analysis condition. Conclusions: The lifetimes of the nucleic acid-binding fluorochromes were altered when binding to different macromolecules under different conditions. Phase-sensitive flow cytometry provided a unique means for simultaneous discrimination and quantification of subcellular macromolecules with one fluorescent probe. The data demonstrated the capabilities for resolving relative cellular DNA and RNA contents based on fluorescence lifetime.