Pulse Width for Particle Sizing

Robert A. Hoffman1

1 BD Biosciences, San Jose, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.23
DOI:  10.1002/0471142956.cy0123s50
Online Posting Date:  October, 2009
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Abstract

The widths of optical pulses in flow cytometry contain information about the size of particles. This size information is independent of many of the factors that affect light scatter as a measure of particle size, and any light scatter or fluorescence signal can be used to measure pulse width. For fluorescence signals, the pulse width can be predicted theoretically for many particle shapes, and quantitative size calibration is possible. To be a meaningful independent parameter, the pulse‐width measurement must be independent of the pulse amplitude. This unit provides protocols for determining the signal range over which amplitude independent pulse‐width measurements can be made and methods for calibrating the pulse‐width measurements to particle diameter. Calibration and application examples are provided and briefly discussed. Curr. Protoc. Cytom. 50:1.23.1‐1.23.17. © 2009 by John Wiley & Sons, Inc.

Keywords: pulse width; flow cytometry; size; fluorescence; light scatter

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determining the Range for Amplitude‐Independent Pulse Width
  • Alternate Protocol 1: Determining the Range for Amplitude‐Independent Pulse Width Using a Multiple Intensity Bead Set
  • Basic Protocol 2: Calibration of the Pulse‐Width Measurement to Particle Diameter
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determining the Range for Amplitude‐Independent Pulse Width

  Materials
  • Beads with diameter in the size range of interest but at least as large as the height of the focused laser beam on the sample stream
  • Diluent
  • Flow cytometer

Alternate Protocol 1: Determining the Range for Amplitude‐Independent Pulse Width Using a Multiple Intensity Bead Set

  Materials
  • Beads of varying fluorescence intensity but the same diameter
  • Diluent
  • Sample container
  • Flow cytometer

Basic Protocol 2: Calibration of the Pulse‐Width Measurement to Particle Diameter

  Materials
  • Uniformly sized beads that cover the size range of interest (it is informative if at least one of the bead diameters is less than the height of the focused laser beam)
  • Flow cytometer
  • Spreadsheet program
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Figures

Videos

Literature Cited

Literature Cited
   Rudolph, N.S., Ohlsson‐Wilhelm, B.M., Leary, J.F., and Rowley, P.T. 1985. Single‐cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells. Cytometry 6:151‐158.
   Sharpless, T.K. and Melamed, M.R. 1976. Estimation of cell size from pulse shape in flow cytofluorometry. J. Histochem. Cytochem. 24:257‐264.
   Steinkamp, J.A. and Crissman, H.A. 1974. Automated analysis of deoxyribonucleic acid, protein and nuclear to cytoplasmic relationships in tumor cells and gynecologic specimens. J. Histochem. Cytochem. 22:616‐621.
   Yang, M‐C.W., Harvey, N.E., Cuchens, M.A., and Buttke, T.M. 1988. Pulse profile analyses of endocytosis in capped B lymphocytes and BCLl cells. Cytometry 9:131‐137.
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