Re: declump those buggers

Posted by Crowell Elizabeth on Mar 23, 2011; 1:16pm
URL: http://imagej.273.s1.nabble.com/declump-those-buggers-tp3685220p3685229.html

Hi Dan, hi Matthias,

This is getting to be a very interesting thread.  I'd like to make a few
comments from a biologist's point-of-view.

Daniel James White a écrit :
> The reason there is not much that just works out of the box is that this is actually a very hard problem.
>  
There is no point making the problem harder than it needs to be.  I
think the majority of ImageJ users want to use ImageJ to get data for
experiments.  Then the problem becomes much simpler, because you simply
need a method that gives you a good estimate of the number of nuclei and
that works equally well on the images from each condition you want to
compare.

> 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.
>  

I agree with that!  Even by eye we cannot be sure how many nuclei there
are exactly.  But this is not the interesting question for the
biologist.  Unless your interest is developing a perfect, miraculous
method to count them, all we really want to know is: Is there a
difference between the control and the other experimental conditions???
If the difference between conditions is much higher than the error rate,
and we can reproduce this difference in multiple experiments, then the
problem is solved for all practical purposes.

> 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.

However, we want to use ImageJ segmentation and analysis to increase the
quality of our data and to SAVE TIME... If we have to repeat the
staining with a new dye, acquire perfect images at high magnification,
and then run deconvolution, certainly we will spend 5 times as much time
on the experiment.  Then it seems faster, actually, to count the nuclei
manually rather than spending so much time to optimize experimental
conditions, even if the data quality is a little lower because subject
to user error.

So, my advice is to determine what precision and accuracy are actually
needed to make a conclusion about the experiment, and then to see if
some simple segmentation methods in ImageJ can give you that accuracy
with the images you already have.
The next time you need to repeat such an experiment, you will know how
to acquire better images for a better automatic analysis.


> 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.
>
>  
Yes, that is a big disadvantage of DAPI staining.  "Remove outliers"
helps a little to reduce the heterogeneous staining.  But what other
dyes are available??  Doesn't Hoescht staining give the same result?

Kind Regards,

Elizabeth Crowell

> 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
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
> [phone] (Work phone at MPI-CBG)
> [phone] (Fax MPI-CBG LMF)
>
> http://www.bioimagexd.net  BioImageXD
> http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
> http://www.chalkie.org.uk                Dan's Homepages
> https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
> dan (at) chalkie.org.uk
> ( white (at) mpi-cbg.de )
>  

--

Elizabeth CROWELL

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