Lasers for Flow Cytometry: Current and Future Trends

Howard M. Shapiro1, William G. Telford2

1 The Center for Microbial Cytometry, West Newton, Massachusetts, 2 Experimental Transplantation and Immunology Branch, National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.9
DOI:  10.1002/cpcy.30
Online Posting Date:  January, 2018
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Lasers are the principal light sources for flow cytometers. Virtually all cytometers are equipped with at least one (and often many more) lasers. This unit covers the various types of lasers available and the qualities that make them suitable or unsuitable for use in flow cytometers. Also included is a discussion of future directions, particularly in the area of tunable laser development. Practical tips are provided for building multilaser cytometer systems. © 2018 by John Wiley & Sons, Inc.

Keywords: flow cytometry; Gaussian beams; illumination optics; lasers

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Table of Contents

  • Introduction
  • Why Lasers are Needed as Light Sources for Flow Cytometry
  • How Lasers Work
  • Lasers Used in Flow Cytometry
  • Lasers in Cytometer Design
  • Laser Safety
  • Future Directions
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

  Dean, P. N. (2001). Confocal microscopy: Principles and practices. Current Protocols in Cytometry, 5, 2.8:2.8.1–2.8.12. doi: 10.1002/0471142956.cy0208s05.
  Doornbos, R. M. P., De Grooth, B. G., Kraan, Y. M., Van Der Poel, C. J., & Greve, J. (1994). Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis. Cytometry, 15, 267–271. doi: 10.1002/cyto.990150312.
  Perfetto, S. P., & Roederer, M. (2007). Increased immunofluorescence sensitivity using 532 nm excitation. Cytometry, 71A, 73–79. doi: 10.1002/cyto.a.20358.
  Shapiro, H. M. (1986). The little laser that could: Applications of low power lasers in clinical flow. Annals of the New York Academy of Sciences, 468, 18‐27. doi: 10.1111/j.1749‐6632.1986.tb42025.x.
  Shapiro, H. M. (2001). Laser beam shaping and spot size. Current Protocols in Cytometry, 1, 1.6:1.6.1–1.6.5. doi: 10.1002/0471142956.cy0106s01.
  Shapiro, H. M. (2004). Scanning laser cytometry. Current Protocols in Cytometry, 28, 2.10:2.10.1–2.10.9. doi: 10.1002/0471142956.cy0210s28.
  Shapiro, H. M., & Perlmutter, N. G. (2001). Violet laser diodes as light sources for cytometry. Cytometry, 44, 133–136. doi: 10.1002/1097‐0320(20010601)44:2<133::AID‐CYTO1092>3.0.CO;2‐S.
  Telford, W. G. (2004). Analysis of UV‐excited fluorochromes by flow cytometry using a near‐UV laser diode. Cytometry, 61A, 9–17. doi: 10.1002/cyto.a.20032.
  Telford, W. G., Babin, S. A., Khorev, S. V., & Rowe, S. H. (2009). Green fiber lasers: An alternative to traditional DPSS green lasers for flow cytometry. Cytometry, 75A, 1031–1039. doi: 10.1002/cyto.a.20790.
  Telford, W. G., Murga, M., Hawley, T., Hawley, R. G., Packard, B. Z., Komoriya, A., … Hubert, C. (2005). DPSS yellow‐green 561 nm lasers for improved fluorochrome detection by flow cytometry. Cytometry, 68A, 36–44. doi: 10.1002/cyto.a.20182.
  Telford, W. G., Subach, F. V., & Verkhusha, V. V. (2009). Supercontinuum white light lasers for flow cytometry. Cytometry, 75A, 450–459. doi: 10.1002/cyto.a.20687.
Key References
  Doornbos et al., 1994. See above.
  Red laser diodes in flow cytometry.
  Harbison, J. P., & Nahory, R. E. (1997). Lasers: Harnessing the atom's light. New York: Scientific American Library.
  Aimed at the interested layman, this book is beautifully illustrated and includes a detailed discussion of the operation of semiconductor lasers.
  Hecht, J. (1992). The Laser Guidebook, (2nd ed.). Blue Ridge Summit, PA: Tab Books (McGraw‐Hill).
  Hecht's books provide substantial technical detail about lasers in a manner accessible to readers without a strong background in physics and engineering.
  Hecht, J. (2008). Understanding lasers: An entry‐level guide, (3rd ed.). New York: Wiley‐IEEE Press.
  Green 532‐nm lasers in flow cytometry.
  Perfetto & Roederer, 2007. See above.
  Red HeNe lasers in flow cytometry.
  Shapiro, 1986. See above.
  This book provides more particulars about specific flow cytometric applications of various lasers than appear here.
  Shapiro, H. M. (2003). Practical flow cytometry, (4th ed.). Hoboken, NJ: Wiley‐Liss.
  First use of violet laser diodes in flow cytometry.
  Shapiro & Perlmutter, 2001. See above.
  Near‐UV laser diodes in flow cytometry.
  Telford, 2004. See above.
  An overview of lasers in flow cytometry.
  Telford, W. G. (2011). Lasers in flow cytometry. Methods in Cell Biology, 102, 375‐410.
  Yellow 561‐nm lasers in flow cytometry.
  Telford et al., 2005. See above.
  Intermediate green‐yellow lasers in flow cytometry.
  Telford, Babin, et al., 2009. See above.
  The best laser sources for the most recent generation of fluorescent proteins.
  Telford, W. G., Hawley, T. S., Subach, F., Verkusha, V. V., & Hawley, R. J. (2012). Flow cytometry of fluorescent proteins (invited review article). Methods, 57, 318–330. doi: 10.1016/j.ymeth.2012.01.003.
  Supercontinuum lasers in flow cytometry.
  Telford, Subach, et al., 2009. See above.
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