My last suggestion is to use the pixel classification program packaged with the latest release of CP, called illastik. It only works on Windows, but can be a powerful tool if regular thresholding and object discrimination doesn’t work. You can find it under the CellProfiler item in the Start Folder menu.
Some quick notes on its use:
[ul][li]When starting a new project (under the Project tab), load in a few representative image files; you shouldn’t need to load them all, and indeed loading too many will cause you memory problems.[/li][li]Under the [i]Classification tab > Select features[/]i, you can try selecting just the color features. Pick the size scale that’s appropriate for your images (I think Huge, Megahuge and Gigahuge will do). Selecting too many will cause memory problems.[/li][li]On the right-hand side, click “Create class” and create the regions that you want to divide the image into; I would make three: blue nuclei, brown nuclei and everything else. You can give these classes the names or colors you want (e.g, call the blue class “Blue”).[/li][li]Select a label and you’ll see that the mouse turns into a cross-hair when waved over the image. Scribble on those parts of the image that correspond to the class selected. You shouldn’t need to be extensive; just a few representative areas.[/li]
[li]Click Classification tab > Train and Predict to try to classify the image areas using the features and labels you’ve provided. You’ll see the displays appear under “Overlays” on the lower right, which you can toggle on or off.I would recommend turn off the Segmentation display since it tends to occlude the overlay.[/li][li]If it doesn’t do well enough, scribble on more areas and then re-train (click Train and Predict)[/li][li]If you’ve happy with the image, select the next image from the upper right, and view the results there. If it doesn’t do well, scribble some regions on the new image.[/li][li]Once you’re happy with the overall result, save the project (Project > Save) and save the classifier (Classification > Export Classifier)[/li][/ul]
At this point, you can use the classifier in CellProfiler by adding the module ClassifyPixels to your pipeline (again, this is only available on Windows). Point the module at the color image and at the classifier (.h5) file, and select the number of the label you want (0 for the 1st label, 1 for the second and so on).
The output of ClassifyPixels is a grayscale image representing the regions which corresponding to the label you want; this can be used as input into IdentifyPrimaryObjects to find the objects of interest. You can use multiple ClassifyPixels modules to highlight the different regions you specify.