Counting nuclear foci - getting strange results

[jswalker]

Hi all,

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.

		   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();