Scanning Laser Cytometry

Howard M. Shapiro1

1 West Newton, null, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.10
DOI:  10.1002/0471142956.cy0210s28
Online Posting Date:  May, 2004
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Abstract

There are many tasks to which flow cytometers are unsuited, e.g., measurement of attached cells, repeated measurements of a single cell over time, and localization of probes in or on cells. Devices such as confocal microscopes and microscope‐based imaging systems allow high‐resolution analysis and provide flexibility, but typically have low sample throughput and often require a high level of interaction with a technically sophisticated operator. Scanning laser cytometers, which can make low‐resolution, multiparameter optical measurements of light scattering and fluorescence similar to those of flow cytometers, provide reasonably high sample throughput and may, depending on the purpose for which they are designed, either allow for considerable flexibility and operator interaction or permit analysis of large numbers of samples without an operator in attendance. This unit presents an overview of sanning laser cytometers, including such topics as the operating principles, data analysis, and commercially available systems and their applications.

Keywords: scanning laser cytometer; Acumen Explorer HTS; CompuCyte Laser Scanning Cytometer; IMAGN 2000; ChemScan RDI

     
 
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Table of Contents

  • Operating Principles
  • Data Analysis
  • Commercially Available Systems and Applications
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

  •   FigureFigure 2.10.1 Schematic of the optics of the LSC laser scanning cytometer (courtesy of CompuCyte Corporation).
  •   FigureFigure 2.10.2 Schematic of the optics of the IMAGN 2000 volumetric capillary cytometer (courtesy of Biometric Imaging).
  •   FigureFigure 2.10.3 Image data represented as oscilloscope traces of serial scan lines from the IMAGN 2000 volumetric capillary cytometer. Fluorescent cells are represented by peaks; the “plateau” on which the peaks lie represents fluorescence from free dye in the capillary. Courtesy of Biometric Imaging.
  •   FigureFigure 2.10.4 Digitized image of propidium‐stained lymphocyte nuclei produced by the LSC, showing threshold, integration, and background contours (see text). Courtesy of CompuCyte Corporation.
  •   FigureFigure 2.10.5 Identification of micronuclei in cells. See text for details. Courtesy of CompuCyte Corporation.
  •   FigureFigure 2.10.6 An xy plot of positions of single nuclei obtained from an image of a skin section analyzed with the LSC, providing a cartoon of the tissue architecture, clearly illustrating the locations of hair follicles. Courtesy of CompuCyte Corporation.

Videos

Literature Cited

Literature Cited
   Barnett, D., Goodfellow, K., Ginnever, J., Granger, V., Whitby, L., and Reilly, J.T. 2001. Low level leucocyte counting: A critical variable in the validation of leucodepleted blood transfusion components as highlighted by an external quality assessment study. Clin. Lab. Haematol. 23:43‐51.
  The author thanks Bala Manian and Kim Mulcahy (formerly of Biometric Imaging) and Kurt Barkalow, Elena Holden, Anne Byrne, Louis Kamentsky, and Ed Luther of CompuCyte Corporation.
   Baudart, J., Coallier, J., Laurent, P., and Prevost, M. 2002. Rapid and sensitive enumeration of viable diluted cells of members of the family enterobacteriaceae in freshwater and drinking water. Appl. Environ. Microbiol. 68:5057‐5063.
   Chapple, P., Prince, H.M., Wall, D., Filshie, R., Haylock, D., Quinn, M., Bretell, M., and Venter, D. 2000. Comparison of three methods of CD34+ cell enumeration in peripheral blood: Dual‐platform ISHAGE protocol versus single‐platform, versus microvolume fluorimetry. Cytotherapy 2:371‐376.
   Darzynkiewicz, Z., Bedner, E., Li, X., Gorczyca, W., and Melamed, M.R. 1999. Laser‐scanning cytometry: A new instrumentation with many applications. Exp. Cell Res. 249:1‐12.
   Darzynkiewicz, Z., Smolewski, P., and Bedner, E. 2001. Use of flow and laser scanning cytometry to study mechanisms regulating cell cycle and controlling cell death. Clin. Lab. Med. 21:857‐873.
   de Roubin, M.‐R., Pharamond, J.‐S., Zanelli, F., Poty, F., Houdart, S., Laurent, F., Drocourt, J.‐L., and Van Poucke, S. 2002. Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters. J. Appl. Microbiol. 93:599‐607;
   Erratum in J. Appl. Microbiol. 2003; 94:158.
   Dietz, L.J., Dubrow, R.S., Manian, B.S., and Sizto, N.L. 1996. Volumetric capillary cytometry: A new method for absolute cell enumeration. Cytometry 23:177‐186.
   Kamentsky, L.A., Burger, D.E., Gershman, R.J., Kamentsky, L.D., and Luther, E. 1997. Slide‐based laser scanning cytometry. Acta Cytol. 41:123‐143.
   Kamentsky, L. A. 2001. Laser scanning cytometry. Methods Cell Biol. 63:51‐87.
   O'Gorman, M.R.G., Gelman, R., Site Investigators, and the NIAID New CD4 Technologies Focus Group. 1997. Inter‐ and intra‐institutional evaluation of automated volumetric capillary cytometry for the quantitation of CD4‐ and CD8‐positive T‐lymphocytes in the peripheral blood of persons infected with human immunodeficiency virus. Clin. Diag. Lab. Immunol. 4:173‐179.
   Seamer, L.C., Bagwell, C.B., Barden, L., Redelman, D., Salzman, G.C., Wood, J.C.S., and Murphy, R.F. 1997. Proposed new data file standard for flow cytometry, version FCS 3.0. Cytometry 28:118‐122.
   Smith, P.J., Wiltshire, M., Davies, S., Patterson, L.H., and Hoy, T. 1999. A novel cell permeant and far red‐fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry. J. Immunol. Methods 229:131‐139.
   Tarnok, A. and Gerstner, A.O. 2002. Clinical applications of laser scanning cytometry. Cytometry 50:133‐143.
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