A user's guide for avoiding errors in absorbance image cytometry: a review with original experimental observations |
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Authors: | Pasquale Chieco Ard Jonker Cinzia Melchiorri Gabriele Vanni and Cornelis J F Van Noorden |
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Institution: | (1) Institute of Oncology, Viale Ercolani 4, 40138 Bologna, Italy;(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;(3) Byk Gulden Italia, Via Giotto 1, Cormano, (Mi), Italy |
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Abstract: | 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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