Capture of Fluorescence Decay Times by Flow Cytometry

Jessica P. Houston1, Mark A. Naivar2, Patrick Jenkins1, James P. Freyer3

1 Department of Chemical Engineering, New Mexico State University, Las Cruces, New Mexico, 2 DarklingX, Los Alamos, New Mexico, 3 National Flow Cytometry Resource, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.25
DOI:  10.1002/0471142956.cy0125s59
Online Posting Date:  January, 2012
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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.

Keywords: fluorescence lifetime; flow cytometry; fluorescence decay kinetics; frequency‐domain; phase‐sensitive detection; digital data systems

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Table of Contents

  • Introduction
  • Basic Protocol 1: Conventional Analog Analysis for Single Lifetime Acquisition
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Conventional Analog Analysis for Single Lifetime Acquisition

  • Bead and/or cell samples (scatter‐only microspheres, calibration fluorescence microspheres, and unknown sample)
  • External laser modulator (electro‐optic modulator device or acousto‐optic modulator device, e.g., Conoptics, model 350‐105‐1, KD*P series electro‐optic modulator with model 200 driver)
  • Radio‐frequency signal synthesizer to drive modulator and provide a mixing signal (e.g., Tektronix, model AFG 3102 function generator)
  • Flow cytometer with at least two photomultiplier tubes with rapid (≤2 nsec) rise time specifications
  • High‐speed transimpedance preamplifiers (0 ≥ bandwidth ≥ 10 MHz, or hundreds of MHz range) for the PMTs used for modulation detection
  • Oscilloscope
  • Delay line with nanosecond resolution and manual adjustment (or other device capable of phase‐shifting an analog sinusoidal signal)
  • High‐frequency signal multipliers (two required)
  • Quadrature hybrid device for creating in‐phase carrier wave and phase‐quadrature, or 90°, out‐of‐phase carrier wave
  • Low‐pass frequency filter (∼100 kHz bandwidth to reduce high frequency)
  • Analog ratio module device (capable of dividing two analog pulse waveforms)
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Literature Cited

Literature Cited
   Beisker, W. and Klocke, A. 1997. Fluorescence lifetime meaurements in flow cytometry. Proc. SPIE Int. Soc. Opt. Eng. 2982:436‐445.
   Bores Signal Processing. FFT Window Functions (n.d) from Surrey, U.K.
   Cui, H.H., Valdez, J.G., Steinkamp, J.A., and Crissman, H.A. 2003. Fluorescence lifetime‐based discrimination and quantification of cellular DNA and RNA with phase‐sensitive flow cytometry. Cytometry A 52:46‐55.
   Deka, C. and Steinkamp, J.A. 1996. Time‐resolved fluorescence‐decay measurement and analysis on single cells by flow cytometry. Appl. Optics 35:4481a7hyphen;4489.
   Deka, C., Sklar, L., and Steinkamp, J.A. 1994. Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique. Cytometry 17:94‐101.
   Deka, C., Cram, L.S., Habbersett, R., Martin, J.C., Sklar, L.A., and Steinkamp, J.A. 1995. Simultaneous dual‐frequency phase‐sensitive flow cytometric measurements for rapid identification of heterogeneous fluorescence decays in fluorochrome‐labeled cells and particles. Cytometry 21:318‐328.
   Deka, C., Lehnert, B.E., Lehnert, N.M., Jones, G.M., Sklar, L.A., and Steinkamp, J.A. 1996. Analysis of fluorescence lifetime and quenching of FITC‐conjugated antibodies on cells by phase‐sensitive flow cytometry. Cytometry 25:271‐279.
   Houston, J.P., Naivar, M.A., Martin, J.C., Goddard, G., Carpenter, S., Mourant, J.R., and Freyer, J.P., 2008. Endogenous fluorescence lifetime of viable cells by flow cytometry. Proc. SPIE Int. Soc. Opt. Eng. 6859:68590T‐68590T8.
   Houston, J.P., Naivar, M.A., and Freyer, J.P. 2010. Digital analysis and sorting of fluorescence lifetime by flow cytometry. Cytometry A 77:861‐872.
   Jin, D., Connally, R., and Piper, J. 2007. Practical time‐gated luminescence flow cytometry. I: Concepts. Cytometry A 71A:783‐796.
   Jin, D., Piper, J., Leif, R.C., Yang, S., Ferrari, B.C., Yuan, J., Wang, G., Vallarino, L.M., and Williams, J.W. 2009. Time‐gated flow cytometry: An ultra high selectivity method to recover ultra‐rare‐event mu‐targets in high‐background biosamples. J. Biomed. Optics 14:024023.
   Keij, J.F. and Steinkamp, J.A. 1998. Flow cytometric characterization and classification of multiple dual‐color fluorescent microspheres using fluorescence lifetime. Cytometry 33:318‐323.
   Lakowicz, J. 2006. Principles of Fluorescence Spectroscopy. Springer, New York.
   Lyons, R.G., 2010. Understanding Digital Signal Processing, Third Edition. Prentice Hall, Upper Saddle River, N.J.
   Naivar, M.A., Parson, J.D., Wilder, M.E., Habbersett, R.C., Edwards, B.S., Sklar, L., Nolan, J.P., Graves, S.W., Martin, J.C., and Jett, J.H. 2007. Open, reconfigurable cytometric acquisition system: ORCAS. Cytometry A 71:915‐924.
   Pinsky, B.G., Ladasky, J.J., Lakowicz, J.R., Berndt, K., and Hoffman, R.A. 1993. Phase‐resolved fluorescence lifetime measurements for flow cytometry. Cytometry 14:123‐135.
   Sailer, B.L., Nastasi, A.J., Valdez, J.G., Steinkamp, J.A., and Crissman, H.A. 1996. Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide. Cytometry 25:164‐172.
   Sailer, B.L., Nastasi, A.J., Valdez, J.G., Steinkamp, J.A., and Crissman, H.A. 1997. Differential effects of deuterium oxide on the fluorescence lifetimes and intensities of dyes with different modes of binding to DNA. J. Histochem. Cytochem. 45:165‐175.
   Sailer, B.L., Steinkamp, J.A., and Crissman, H.A. 1998a. Flow cytometric fluorescence lifetime analysis of DNA‐binding probes. Eur. J. Histochem. 42:19‐27.
   Sailer, B.L., Valdez, J.G., Steinkamp, J.A., and Crissman, H.A. 1998b. Apoptosis induced with different cycle‐perturbing agents produces differential changes in the fluorescence lifetime of DNA‐bound ethidium bromide. Cytometry 31:208‐216.
   Steinkamp, J.A. 2000. Time‐resolved fluorescence measurements. Curr. Protoc. Cytom. 11:1.15.1‐1.15.16.
   Steinkamp, J.A. and Keij, J.F. 1999. Fluorescence intensity and lifetime measurement of free and particle‐bound fluorophore in a sample stream by phase‐sensitive flow cytometry. Rev. Sci. Instrum. 70:4682‐4688.
   Steinkamp, J.A., and Crissman, H.A. 1993. Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase‐sensitive flow cytometry. Cytometry 14:210‐216.
   Steinkamp, J.A. and Parson, J.D. 2001. Flow cytometric, time‐resolved measurements by frequency heterodyning of fluorescence emission signals. Proc. SPIE Int. Soc. Opt. Eng. 4260:166‐174.
   Steinkamp, J.A., Yoshida, T.M., and Martin, J.C. 1993. Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome‐labeled cells/particles by phase‐sensitive detection. Rev. Sci. Instrum. 64:3440‐3450.
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