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Analytical Cellular Pathology
Volume 24, Issue 2-3, Pages 101-111

Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

Carolina Wählby,1 Joakim Lindblad,1 Mikael Vondrus,1 Ewert Bengtsson,1 and Lennart Björkesten2

1Centre for Image Analysis at Uppsala University, Uppsala, Sweden
2Amersham Pharmacia Biotech, Uppsala, Sweden

Received 2 August 2001; Accepted 24 June 2002

Copyright © 2002 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.


Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre‐processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical analysis of a number of shape descriptive features. Objects that have features that differ to that of correctly segmented single cells can be further processed by a splitting step. By statistical analysis we therefore get a feedback system for separation of clustered cells. After the segmentation is completed, the quality of the final segmentation is evaluated. By training the algorithm on a representative set of training images, the algorithm is made fully automatic for subsequent images created under similar conditions. Automatic cytoplasm segmentation was tested on CHO‐cells stained with calcein. The fully automatic method showed between 89% and 97% correct segmentation as compared to manual segmentation.