quantification of fluorescent signal per a cell

[Junsung LEE]

I want to quantify fluorescent signal (punctiform, like cellular vesicles) in one cell, but I don't know how. I have tried a few things as follows (but all of them were not successful) :


1. first, I selected one cell with 'polygon selections tool', and tried to analyse fluorescent signal with 'RGB profile (Plugins>Graphics>RGB profile)'. But I failed because 'RGB profile' is working only with 'line or rectangular selections tool'. I can apply 'rectangular selections tool' in some cases, but when cells are overlapped partially, it is not applicable.

> is there any ways to make 'RGB profile' work with 'polygon selections tool' or 'freehand selections tool'?


2. second, I marked fluorescent signals (they are dot-shaped, as I said above) with 'multi-point selections tool' one by one, and analysed them with 'measure (analyse>measure (ctrl+M)'. This method is working but marking many fluorescent dots one by one is really hard.

> I really wonder how to select fluorescent signals (dots) automatically. Once I designate one dot as signal, it is possible to select dots automatically, I think, but I don't know how.


I will be appreciated if you tell me how. If appropriate method is exist except what I said above, please don't hesitate to   let me know. Please refer to attached image file.

Thanks in advance.


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[Stoyan Pavlov]

Hi Lee,
I had similar problem several years ago, so i think i may be able to give
you some advice.
 I had also to run measurements inside multiple cells (RoIs) so i had to
develop my own macro. It is crude but it did the job for me so if you can
you might try and modify it to your needs. This macro is run after your
images are segmented, the RoIs are generated and loaded in the RoIManager.

// This Macro counts particles and measures particle covered area inside
specific Roi and otputs the numbers //to TAB separated Log window which
after saving can be imported to excel.
print ("RoiNumber","    ","SliceLabel","    ","RoIName","    ","Count","
","RoIArea","    ","RoIPerimeter","    "," ParticlesCoveredArea","
","AreaFraction") ;
setAutoThreshold("Default dark");
 n = roiManager("count");
        for (i=0; i<n; i++)  {
        roiManager("select", i);
        run("Set Measurements...", "area perimeter redirect=None
decimal=4");
    run("Measure");
    r=getResult("Area");
    u=getResult("Perim.");
run("Set Measurements...", "area area_fraction limit display redirect=None
decimal=4");
run("Measure");
s=getResult("Area");
t=getResult("%Area");
run("Analyze Particles...", "size=0-20 circularity=0.00-1.00 show=Nothing
display clear ");
    m=nResults;
     q=getInfo("slice.label");
    print (i,"    ",q,"    ",call("ij.plugin.frame.RoiManager.getName",
i),"    ",m,"    ",r,"    ",u,"    ",s,"    ",t) ;
}



As to how to segment your images there are many plugins and explanations,
depending on your needs and goals. One of the easiest and quickest is to
simply threshold them. As it wan't work perfectly for your images you need
to try different segmentation strategies and see which one is the best for
you. For example there is a plugin for FIJI that does something similar to
what you were asking for - Trainable Segmentation. It can be found here
http://fiji.sc/Trainable_Segmentation_Plugin
Don't worry once you choose your method it can easily be automated using
the macro recording functions of ImageJ (or Fiji). For example there is one
very nice  macro that lets you process entire folders and it can be found
here (http://rsb.info.nih.gov/ij/macros/BatchProcessFolders.txt).

Hope this can help you a little bit. If you have further questions ask them
as there are many people here that can help you much more than me.



2013/3/13 Junsung LEE <[hidden email]>

> I want to quantify fluorescent signal (punctiform, like cellular vesicles)
> in one cell, but I don't know how. I have tried a few things as follows
> (but all of them were not successful) :
>
>
> 1. first, I selected one cell with 'polygon selections tool', and tried to
> analyse fluorescent signal with 'RGB profile (Plugins>Graphics>RGB
> profile)'. But I failed because 'RGB profile' is working only with 'line or
> rectangular selections tool'. I can apply 'rectangular selections tool' in
> some cases, but when cells are overlapped partially, it is not applicable.
>
> > is there any ways to make 'RGB profile' work with 'polygon selections
> tool' or 'freehand selections tool'?
>
>
> 2. second, I marked fluorescent signals (they are dot-shaped, as I said
> above) with 'multi-point selections tool' one by one, and analysed them
> with 'measure (analyse>measure (ctrl+M)'. This method is working but
> marking many fluorescent dots one by one is really hard.
>
> > I really wonder how to select fluorescent signals (dots) automatically.
> Once I designate one dot as signal, it is possible to select dots
> automatically, I think, but I don't know how.
>
>
> I will be appreciated if you tell me how. If appropriate method is exist
> except what I said above, please don't hesitate to   let me know. Please
> refer to attached image file.
>
> Thanks in advance.
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

... [show rest of quote]

--
Dr. Stoyan P. Pavlov, MD, PhD
Departament of Anatomy, Histology and Embryology
Medical University "Prof. Dr. Paraskev Stoyanov", Varna
Prof. Marin Drinov Str.55
9002 Varna
 Bulgaria
Tel: +359 (0) 52 - 677 - 086
e-mail: [hidden email]

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[Shall, Sydney]

On 13/03/2013 02:22, Junsung LEE wrote:

> I want to quantify fluorescent signal (punctiform, like cellular vesicles) in one cell, but I don't know how. I have tried a few things as follows (but all of them were not successful) :
>
>
> 1. first, I selected one cell with 'polygon selections tool', and tried to analyse fluorescent signal with 'RGB profile (Plugins>Graphics>RGB profile)'. But I failed because 'RGB profile' is working only with 'line or rectangular selections tool'. I can apply 'rectangular selections tool' in some cases, but when cells are overlapped partially, it is not applicable.
>
>> is there any ways to make 'RGB profile' work with 'polygon selections tool' or 'freehand selections tool'?
>
> 2. second, I marked fluorescent signals (they are dot-shaped, as I said above) with 'multi-point selections tool' one by one, and analysed them with 'measure (analyse>measure (ctrl+M)'. This method is working but marking many fluorescent dots one by one is really hard.
>
>> I really wonder how to select fluorescent signals (dots) automatically. Once I designate one dot as signal, it is possible to select dots automatically, I think, but I don't know how.
>
> I will be appreciated if you tell me how. If appropriate method is exist except what I said above, please don't hesitate to   let me know. Please refer to attached image file.
>
> Thanks in advance.
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

... [show rest of quote]

Dear Junsung,
The answer that you need was posted yesterday by myself and someone else.
I copy below the two answers.

Dear Rona,
There is a plug-in for ImageJ crafted by Dr Pawel Znojek, which
essentially aims to do this.
He constructed it for DAPI and a fluorescent  antibody to a DNA repair
protein.
You should be able to adapt this easily for your purposes.
The plugin is available from the following site:
http://www.pzfociez.com/ .
I would recommend that you should add a plugin constructed by Gabriel
Landini, at the University of Birmingham, England, which helps considerably.
This you can find on the ImageJ site.

If you proceed down this path, I should be very grateful to you if you
could let me know how things develop for you.

With best wishes,

Sydney

hi
you can try the color deconvolution way ( landini's plugin)
or the color segmentation,
moreover, is available online a software developed for ki-67 nuclear staining
quantificationhttp://breast-cancer-research.com/content/12/4/R56
maybe at 40 x magnification it could work.
Let me know
Good luck

carlo  bologna Italy


With best wishes,
Sydney

--
Professor Sydney Shall,
Department of Haematological Medicine,
King's College London,
Medical School,
123 Coldharbour Lane,
LONDON SE5 9NU,
Tel & Fax: [phone],
E-Mail: sydney.shall,
[correspondents outside the College should add; @kcl.ac.uk]
www.kcl.ac.uk

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