Capture of Fluorescence Decay Times by Flow Cytometry

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead‐based assays has persisted among the greater cytometry community. In this unit, new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow cytometry are described. This unit provides protocols and a Commentary section describing a most comprehensive discourse on acquiring the fluorescence lifetime with frequency‐domain methods. This unit covers (1) standard fluorescence lifetime acquisition (protocol‐based) with frequency‐modulated laser excitation, (2) digital frequency‐domain cytometry analyses, and (3) interfacing fluorescence lifetime measurements onto sorting systems. This unit also discusses how digital methods are used for aliasing to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi‐exponential decay extraction. Curr. Protoc. Cytom . 59:1.25.1‐1.25.21. © 2012 by John Wiley & Sons, Inc.