Characterization of Flow Cytometer Instrument Sensitivity

Robert A. Hoffman1, James C.S. Wood2

1 BD Biosciences, San Jose, California, 2 Wake Forest University School of Medicine, Winston‐Salem, North Carolina
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.20
DOI:  10.1002/0471142956.cy0120s40
Online Posting Date:  April, 2007
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Abstract

Fluorescence sensitivity, measured in terms of resolution, allows the researcher to more accurately answer the question of how dim a cell can be and still be resolvable from another population. This measure focuses on the width, i.e., the standard deviation, of the population distributions and not on the location, i.e., the mean intensity, of populations on a histogram scale relative to the background. To determine the fluorescence sensitivity in terms of the resolution, both the detection efficiency, Q, and optical background, B, need to be measured. Both factors affect the ability to resolve dimly fluorescent subpopulations, and both factors are required to uniquely characterize the performance of a flow cytometer. Using Q and B, it is possible to determine the minimum fluorescence intensity that is resolvable from the background or from another population. This unit describes a practical and robust approach to measure the critical factors that determine the ability of a flow cytometer to analyze dimly fluorescent particles.

Keywords: fluorescence; sensitivity; resolution; Q; detection efficiency; background

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Flow Cytometer Instrument Detection Efficiency (Q) and Optical Background (B)
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

Videos

Literature Cited

Literature Cited
   Chase, E.S. and Hoffman, R.A. 1998. Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity. Cytometry 33:267‐279.
   Schwartz, A., Wang, L., Early, E., Gaigalas, A.K., Zhang, Y.‐Z., Marti, G.E., and Vogt, R.F. 2002. Quantitating fluorescence intensity from fluorophore: The definition of MESF assignment. J. Res. Natl. Inst. Stand. Technol. 107:83‐91.
   Shapiro, H.M. 2003. Practical Flow Cytometry, 4th edition. John Wiley & Sons, Hoboken, N.J.
   Steen, H.B. 1992. Noise, sensitivity, and resolution of flow cytometers. Cytometry 13:822‐830.
   Wood, J.C.S. 1998. Fundamental flow cytometer properties governing sensitivity and resolution. Cytometry 33:260‐266.
   Wood, J.C.S. and Hoffman, R.A. 1998. Evaluating fluorescence sensitivity on flow cytometers: An overview. Cytometry 33:256‐259.
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