Flow cytometric fluorescence lifetime analysis of DNA-binding probes

A new dimension has been added to multiparameter flow cytometric analysis t hrough the recent development of techniques for rapidly measuring the fluor escence lifetime of probes bound to single cells. The lifetime measurements are made by phase-sensitive detection techniques in a flow cytometer (FCM) that also analyzes fluorescence intensity and other optical properties of stained cells. These lifetime assays have potential for elucidating the mic roenvironment of the interaction of fluorochrome probes and subcellular tar get molecules. Alterations in the lifetime of DNA probes have been observed in cells in different phases of the cell cycle, in different cell types, i n differentiating cells, and in apoptotic cells with damaged chromatin. Lif etime differences noted also for intercalating dyes bound to DNA and dsRNA, indicated modifications in the modes of binding and provide the potential for analyzing both corformational states and nucleic acid metabolism. Futur e developments in the technology will provide multiple lifetime assays and thereby allow for detection and quantitation of selected subcellular probe- complexes with different lifetime signatures. These novel assays will expan d the applications for quantitative studies on the binding of various chemi cal agents to DNA and other molecular targets in cells, and further improve methods for rapid screening of chemotherapeutic agents or environmentally toxic compounds.