title: Capture of Fluorescence Decay Times by Flow Cytometry.
abstract: 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, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction.
authors.affiliation: Department of Chemical Engineering, MSC 3805, New Mexico State University, P.O. Box 30001, Las Cruces, NM 88003-8001, (575) 646-5563, fax: (575) 646-7706.
authors.forename: Jessica P
authors.initials: JP
authors.lastname: Houston
authors.affiliation: DarklingX, 1199 45 Street, Los Alamos, NM 87544.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Naivar
authors.affiliation: Department of Chemical Engineering, MSC 3805, New Mexico State University, P.O. Box 30001, Las Cruces, NM 88003-8001, (575) 646-5563, fax: (575) 646-7706.
authors.forename: Patrick
authors.initials: P
authors.lastname: Jenkins
authors.affiliation: National Flow Cytometry Resource, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545.
authors.forename: James P
authors.initials: JP
authors.lastname: Freyer
keywords: digital data systems; flow cytometry; fluorescence decay kinetics; fluorescence lifetime; frequency-domain; phase-sensitive detection