Comparison of stains for image segmentation and measurement of nuclear parameters by computerised image analysis...

Nuclear measurements using image analysis largely depend upon the quality of the image presented for digitization. To investigate the effects of nuclear stains on image segmentation of nuclei, serial sections of kidney were stained by eleven different methods and presented to an IBAS 2000 interactive image analysis system (Kontron Bildanalyse) via a Zeiss IIIRS microscope at x 800 magnification and a Siemens K30 video camera. Digitised grey level images of each field were processed by an interactive technique and by an automatic segmentation procedure (thresholding). Nuclear areas were measured by each method and the results compared. We conclude that of the stains assessed the uncounterstained haematoxylins offer the best image segmentation for nuclear measurements. Thresholding techniques are suitable for performing measurements using these stains, particularly when additional interactive techniques are used to reject unwanted structures and to separate overlapping nuclei after segmentation. Comparable areas stained with five of the stains were studied to see if the staining techniques themselves affected nuclear area. Our results show that the use of different stains will substantially affect measurements of nuclear dimensions.