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Analytical Cellular Pathology
Volume 15, Issue 3, Pages 145-156

A New Method for Segmentation of Colour Images Applied to Immunohistochemically Stained Cell Nuclei

Petter Ranefall,1 Lars Egevad,2 Bo Nordin,1 and Ewert Bengtsson1

1Centre for Image Analysis, Uppsala University, Sweden
2Department of Pathology, Uppsala University, Sweden

Received 29 November 1996; Revised 1 July 1997; Accepted 7 October 1997

Copyright © 1997 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


A new method for segmenting images of immunohistochemically stained cell nuclei is presented. The aim is to distinguish between cell nuclei with a positive staining reaction and other cell nuclei, and to make it possible to quantify the reaction. First, a new supervised algorithm for creating a pixel classifier is applied to an image that is typical for the sample. The training phase of the classifier is very user friendly since only a few typical pixels for each class need to be selected. The classifier is robust in that it is non‐parametric and has a built‐in metric that adapts to the colour space. After the training the classifier can be applied to all images from the same staining session. Then, all pixels classified as belonging to nuclei of cells are grouped into individual nuclei through a watershed segmentation and connected component labelling algorithm. This algorithm also separates touching nuclei. Finally, the nuclei are classified according to their fraction of positive pixels.