Posted by
Daniel James White on
Mar 23, 2011; 10:37am
URL: http://imagej.273.s1.nabble.com/declump-those-buggers-tp3685220p3685228.html
Hi Matthias
On Mar 23, 2011, at 5:00 AM, IMAGEJ automatic digest system wrote:
> Date: Mon, 21 Mar 2011 13:19:39 -0700
> From: Matthias Kirsch <
[hidden email]>
> Subject: declump those buggers
>
> dear all,
>
> 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 images is obsolete.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 presented in a graph with freely
> chosen measurement as parameters as axes. Then one could click on the
> individual points in the graph and see the corresponding cell in the image
> and then decide whether to keep or discaed it. This should also work the
> other way round, i.e. click on a cell in the image and see the corresponding
> point in the graph. Similarily, it would be most helpful to be able do
> somethig like gating in FACS, i.e. set limits to certain parameters and see,
> which cells fulill them.
>
> I know that this is asking a lot, but I think providing this kind of
> routines in an easy to use plugin would make ImageJ even more attractive and
> competitive than it already is. Of course this applies only for
> 'Non-Cracks'.
The reason there is not much that just works out of the box is that this is actually a very hard problem.
THe one that solves it in a robust and generally applicable way will get very famous.
The DNA staining is non uniform, and the nuclei are very close, so the feature you really need to get is the space between them,
and in your images, that resolution simply isn't there!
In amy cases even by eye, with a clever human brain , its hard to say it there are 1 or 2 nuclei in a certain blob.
Clever tricks like watershed separtation only get you so far.
here is a pipeline that sometimes works
0) maybe forget dapi staining.. it doesnt give you the info you actually want... where is the nucleus.
find a stain that gives the spatial info you actually want.
1) Get as high resolution, blur free images as possible so you can see the gaps between the nuclei (deconvolution might help increase contrast -thats a whole story in itself)
2) find a method to smooth out the blobbyness of the nuclear staining.... without losing the edges... maybe anisotropic diffusion or some other fancy filtersing
3) compute the gradient image or some difference of gaussian or whatever feature extraction
4) use that for the object selection... fix the errors somehow.
>
> Any help would be welcome. Further questions alnosr guaranteed.
>
sorry there is nothing specific...
but start by assessing if the info you want is even given by the dapi staining you are using.
Its often assumed that it is, since it kinda looks like it does, but in fact it is often not the case.
DAPI does not stain "the nucleus"
it stains the chromatin which is heterogeneously distributed in the nucleus, and also stains the cytoplasm a little.
cheers
Dan
> Cheers
>
> P.S.: Can only insert jpeg images
> Matthias
http://imagej.588099.n2.nabble.com/file/n6193826/RGB_%28blue%29.jpg
Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
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