I'm following the protocol outlined here: http://microscopy.duke.edu/HOWTO/countfoci.html
And the commands I'm using are enclosed below.
My problem is that I'm working on massive images (25k x 17k pixels) and sometimes I get accurate values but sometimes I get what I see below. Going by results table (and by RawIntDen/255 to get the actual number of nuclear foci), the cluster highlighted has around 60,000 nuclei! ...which is not the case as you can see by a quick visual examination.
Any idea why this is or what I can do about it?
I've tried re-binarizing the image in the step immediately before measuring. That didn't work. I get this problem whether I do the commands manually or whether I do it via the macro. Any other ideas?
Thanks in advance.
Link to picture: https://www.dropbox.com/s/b7p6wkijf5smlpy/photo%20aug%2009%2C%204%2054%2021%20pm.jpg?dl=0
run("8-bit");
run("Auto Threshold", "method=Triangle white setthreshold show");
run("Convert to Mask");
run("Fill Holes");
run("Dilate");
run("Dilate");
run("Analyze Particles...", "size=50-Infinity display clear summarize add in_situ");
wait(30000);
run("Revert");
run("Find Maxima...", "noise=7 output=[Single Points] exclude");
run("ROI Manager...");
roiManager("Show None");
roiManager("Show All");
run("Set Measurements...", "area mean min integrated redirect=None decimal=3");
roiManager("Measure");
wait(30000);
roiManager("Save", path + ".zip");
saveAs("Results", path + ".xls");
close();
your pictures seem to be quite big in Pixels. Your defined particle size is "50 - infinity". I think this is too small, try something bigger than 50. hope that solves the Problem.
P.S.: We've tried to cope with similar problems also using this algorithm, perhaps that's going to help you: http://focinator.oeck.de/.