How to automatically count immune cells from TIFF file

I have the following TIFF image downloadable as [TIFF data (zipped)] (https://dl.dropboxusercontent.com/u/16295340/04_709_channel-3.tif.zip) (160MB).

It looks like this:

I assign the arrow there to indicate all the little the white specs (neutrophils) which I want to count.
What’s the best way to adjust the image and count those cells automatically?

Neutrophils can be a bit tricky based on their granularity… so I would first apply a Gaussian - to try to deal with that. In any case - you will have trouble delineating them when these cells cluster.

I just ran these simple steps as a starting-off point for you - but for sure, other experts on here will have more refined methods I’m sure. For example - I set the Otsu threshold to grab all pixels above ~4900-5600 (should play with this a bit yourself to see - or Auto Threshold methods) and the 50-200 range may not be the best size cut-off - you’ll have to play and bit to see what works best.

run("Gaussian Blur...", "sigma=1");
setAutoThreshold("Default dark");
//run("Threshold...");
setAutoThreshold("Otsu dark");
run("Convert to Mask");
run("Watershed");
run("Analyze Particles...", "size=50-200 display exclude add");  
roiManager("Show All");
roiManager("Show All with labels");

You might also find this workshop helpful:
http://imagej.github.io/presentations/fiji-segmentation/#/

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