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A user's guide for avoiding errors in absorbance image cytometry: a review with original experimental observations

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Summary

The sources of errors which may occur when cytophotometric analysis is performed with video microscopy using a charged-coupled device (CCD) camera and image analysis are reviewed. The importance of these errors in practice has been tested, and ways of minimizing or avoiding them are described. Many of these sources of error are known from scanning and integrating cytophotometry; they include the use of white instead of monochromatic light, the distribution error, glare, diffraction, shading distortion, and inadequate depth of field. Sources of errors specifically linked with video microscopy or image analysis are highlighted as well; these errors include blooming, limited dynamic range of grey levels, non-linear responses of the camera, contrast transfer, photon noise, dark current, read-out noise, fixed scene noise and spatial calibration. Glare, contrast transfer, fixed scene noise, depth of field and spatial calibration seem to be the most serious sources of errors when measurements are not carried out correctly. We include a table summarizing all the errors discussed in this review and procedures for avoiding them. It can be concluded that if accurate calibration steps are performed and proper guidelines followed, image cytometry can be applied safely for quantifying amounts of chromophore per cell or per unit volume of tissue in sections, even when relatively simple and inexpensive instrumentation is being used.

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Authors and Affiliations

  1. Institute of Oncology, Viale Ercolani 4, 40138, Bologna, Italy

    Pasquale Chieco & Cinzia Melchiorri

  2. Laboratories of Cell Biology and Histology and of Anatomy and Embryology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands

    Ard Jonker & Cornelis J. F. Van Noorden

  3. Byk Gulden Italia, Via Giotto 1, Cormano, (Mi), Italy

    Gabriele Vanni

Authors
  1. Pasquale Chieco
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  2. Ard Jonker
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  3. Cinzia Melchiorri
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  4. Gabriele Vanni
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  5. Cornelis J. F. Van Noorden
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Chieco, P., Jonker, A., Melchiorri, C. et al. A user's guide for avoiding errors in absorbance image cytometry: a review with original experimental observations. Histochem J 26, 1–19 (1994). https://doi.org/10.1007/BF02388388

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  • DOI: https://doi.org/10.1007/BF02388388

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