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26 June 1992 Analysis of three-dimensional images in quantitative microscopy
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In bright field microscopy, quantitative analysis of acquired images is customarily performed using the `best' image. Since an image with sufficient detail and clarity is required for consistent classification and discrimination of objects in the image, the image with higher magnification is commonly chosen for the analysis, which generally corresponds to lower focal depths. The objectively determined `best' focus level, although optimal for the extraction of some features from the chosen objects, may not correspond to the best focal level for the extraction of some other features. To obtain tighter distribution of all features, we have been searching for a method which employs analysis of images acquired at different focal planes. In this work, we analyzed images of stained cervical cells using three different approaches. In the first approach, different features were extracted from images taken at different focal planes. In the second approach, we used simultaneously all the in-focus and out-of-focus information from the images to reconstruct the focussed images at various focal planes. In the third approach, the in-focus three-dimensional scene was compressed to two dimensions to simulate an image taken from a system with a very large depth of focus. The latter method reduced the data storage size and simplified subsequent scene analysis. The advantages and disadvantages of the above approaches are discussed.
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Steven S. S. Poon, Rabab K. Ward, and Branko Palcic "Analysis of three-dimensional images in quantitative microscopy", Proc. SPIE 1660, Biomedical Image Processing and Three-Dimensional Microscopy, (26 June 1992);

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