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Basic strategies for valid cytometry using image analysis

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Abstract

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetics in difficult imaging situations, such as low fluorescence signals and high absorbance signals.

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Authors and Affiliations

  1. Laboratory of Cell Biology and Histology and Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PB 22700, 1100 DE, Amsterdam, The Netherlands

    A. Jonker

  2. Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, PB 22700, 1100 DE, Amsterdam, The Netherlands

    W. J. C. Geerts, A. F. M. Moorman & W. H. Lamers

  3. Institute of Oncology ‘Felice Addarii’, Viale Ercolani 4, 40138, Bologna, Italy

    P. Chieco

  4. Laboratory of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, PB 22700, 1100 DE, Amsterdam, The Netherlands

    C. J. F. Van Noorden

Authors
  1. A. Jonker
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  2. W. J. C. Geerts
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  3. P. Chieco
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  4. A. F. M. Moorman
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  5. W. H. Lamers
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  6. C. J. F. Van Noorden
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Jonker, A., Geerts, W.J.C., Chieco, P. et al. Basic strategies for valid cytometry using image analysis. J Mol Hist 29, 347–364 (1997). https://doi.org/10.1023/A:1026434816947

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