This a stained cardiac muscle for CD3 positive cells and I need to count the number of cells/square mm.The cells are the intense brown dots. I am using Fiji plugins.
Firstly how can i make the threshold to count automatically the cells, since the intense of brown color is not strong.
I would generally do this using other software (simply due to familiarity) so I do not have any specifics, but your workflow should be to:
1 create a “tissue” object or ROI first (in cellprofiler that might be a primary object?), and then
2 find objects over a certain color deconvolved threshold within that tissue object. The count of objects divided by the area of the tissue should get you what you want.
2b. you may not need to use color deconvolution if your only staining is DAB, as long as most of your images do not have dark artifacts of other colors.
to elaborate on @Research_Associate’s comment on how to potentially implement this strategy in Fiji:
Defining the ROI of the tissue (to measure the area) could work by converting the image to 8-bit, applying a Median filter and thresholding (this is using the auto-Threshold “Minimum”).
To count the CD3-positive cells you can try to “Find Maxima”. If the darkest objects in your images are your cells, you usually have a good chance of finding a prominence-threshold that works well.
The detected cells would then look similar to this:
If there are other objects of different colour in your image, you might need to run a Colour deconvolution first.
Or as a Macro-Version that returns a measure of the tissue-area and the count of cells for this image.
run("Duplicate...", " "); run("8-bit"); run("Median...", "radius=5"); setAutoThreshold("Minimum"); run("Create Selection"); run("Measure"); run("Find Maxima...", "prominence=50 light output=Count");
hope this helps a little bit.