Re: declump those buggers

Posted by Gabriel Landini on Mar 23, 2011; 9:44am
URL: http://imagej.273.s1.nabble.com/declump-those-buggers-tp3685220p3685225.html

> Matthias Kirsch a écrit :
> I am struggling with automatic image analysis. One of the first stumbling
> point seems to be segmenting DAPI-stained nuclei in fluorescence images.
> If this is not working reliably, the whole point of processing large
> numbers of images is obsolete. Attached are examples of such clumps
> (there are many more). If segmentation cannot separate them, it would be
> helpful to have at least some way of eliminating false results by just
> clicking on them, or even better, have all identified particles

Without knowing exactly what you tried, saying that "it does not work" leaves
a lot to be explained.

There are many useful procedures that use greyscale morphology (Pierre
Soille's book is an excellent source) and I would suggest looking at the book.
I have implemented quite a few of the procedures described in the Morphology
collection).

For example, I took your image, smoothed with a gaussian filter or radius 2,
then applied the procedure known as White Hat by Reconstruction with a radius
of size 6. This is the macro procedure:

// GreyWhiteTopHatByReconstruction
// G. Landini at bham. ac. uk.
// 1/Oct/2010
setBatchMode(true);
a=getTitle();
run("Duplicate...", "title=_seed");
run("Minimum...", "radius=6");
run("GreyscaleReconstruct ", "mask="+a+" seed=_seed");
imageCalculator("Subtract create", a,"_seed");
selectWindow("Result of "+a);
rename("WhiteTopHatReconstructed");
setBatchMode(false);

For this to work you will need the Morphology plugins installed (specifically
the GreyscaleReconstruct). If you threshold the result image >0 you will get
lots of blobs, including those which mark the nuclei, most of these are
separated. If not separated, you can apply the watershed separation to this
binary image.

You then threshold the smoothed original and use that binary image as a "mask"
and the result of the top hat above as a "seed" to dilate the seeds without
merging inside the mask. There are 2 or 3 procedures that can do this:
InfluenceZones, DilateNoMerge8 or BinaryGeodesicDilateNoMerge8. The first one
will not work for this example, the 2nd might do, they are both in the
Morphology plugins in my site. The 3rd one (the one I used in the example) I
can send it to those interested as I had no time to upload it yet.

Then you AND the original and the result of the last "dilation without
merging". That would preserve those dilated seeds that existed in the original
thresholded image. Since the seeds were separated, the dilation without
merging preserves the individual seeds.

The ROIS of the result on top of the original are shown in the image attached.
Not perfect, but it would narrow down the search for those clusters impossible
to separate.

Regards

Gabriel