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. 2016 Sep 20;265(2):185–195. doi: 10.1111/jmi.12474

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© 2016 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Automated global thresholding. (A) A greyscale haematoxylin stained image of a monolayer of H400 keratinocytes cell line, grown on glass (magnification ×20, field width 168 μm), preprocessed with a Gaussian filter of radius 1. (B) Image gradient of (A), computed with the Beucher morphological gradient and displayed as pseudo‐colour, for clarity purposes. (C) Thresholded (overlay in blue) image obtained using the value L at which G _Total (L) is a maximum (L = 200). (D) Result of using the value L at which G _Average (L) shows a maximum (L = 180). Note how, in this particular image, (D) corresponds faithfully to the extent of the nuclei depicted in (A). (E) Plots of G _Total (L) and G _Average (L) as a function of threshold L for (A).