I am a relatively new user trying to work with FIJI to upload .scn files of tissue sections which have been stained with two immunofluorescent stains.
I have not been able to import these files and analyze the amount each stain has stained (Blue- all cells and green-some cells). I want to work out what proportion the relative proportion of green cells are to the whole (blue-DAPI) tissue. Importantly I do not want to count actual cell number but rather more the area occupied by each stain.
I have been able to upload .tiff files and work out the area occupied by one channel at a time (and creating separate files for each). I think this is not really the most efficient way and wanted to see if somebody here can help and point me inthe righ direction?
I am unable to upload an example .scn but can attach an image of what the contents looks like. Thank you very much!
you can try the following macro. This is definitely not perfect and I concentrated only on the bright blue regions since I was not sure if the darker blue shadows are out of focus parts, artifacts or if they still belong to the real staining of interst.
original = getTitle(); run("Split Channels"); close(original + " (red)"); selectWindow(original + " (green)"); run("Subtract Background...", "rolling=60"); run("Auto Threshold", "method=Default white"); selectWindow(original + " (blue)"); run("Gaussian Blur...", "sigma=2"); run("Median...", "radius=4"); run("Enhance Contrast...", "saturated=1 normalize"); run("Auto Threshold", "method=Default white"); run("EDM Binary Operations", "iterations=1 operation=dilate"); run("Create Selection"); selectWindow(original + " (green)"); run("Restore Selection"); run("Set Measurements...", "area mean standard modal min centroid center perimeter bounding fit shape feret's integrated median skewness kurtosis area_fraction display redirect=None decimal=3"); run("Measure");
Thus, you will need to optimize the macro but it should serve as a starting point.
In this case you also need the BioVoxxel toolbox because of the EDM dilation function used in the macro. If you prefer to do it without this function you could also include the normal
run("Dilate"); (shouldn’t make a difference in this specific case)
Potential for improvement lies definitely in finding a better extraction threshold or improve the filtering for the blue channel.
Hope it helps.