vesicle distribution in cell population

[CosminM]

Hi All,

I have 2 D images of double stained cells - the cell membrane and some cytoplasmic vesicles – the number of which varies from cell to cell. Each image has 10-30 cells, and both the membrane stain and the vesicle stain can be easily segmented.

I am interested to extract a distribution of the vesicle number inside the individual cells at the cell population level, and to do that in an automatic fashion. I was thinking something along the lines of generating a mask from the membrane stain, registering the position and the outline of the cell area, and then apply that information to the image containing the vesicle stain in order to count and assign the number of vesicles to each cell.

Any ideas on to how to implement this with ImageJ?

Thanks,
Cosmin

[Rainer Kohler-2]

Hi,

Try CellProfiler
http://www.cellprofiler.org/

It should do the job .

Rainer


Cosmin Mihai wrote:

> Hi All,
>
> I have 2 D images of double stained cells - the cell membrane and some cytoplasmic vesicles – the number of which varies from cell to cell. Each image has 10-30 cells, and both the membrane stain and the vesicle stain can be easily segmented.
>
> I am interested to extract a distribution of the vesicle number inside the individual cells at the cell population level, and to do that in an automatic fashion. I was thinking something along the lines of generating a mask from the membrane stain, registering the position and the outline of the cell area, and then apply that information to the image containing the vesicle stain in order to count and assign the number of vesicles to each cell.
>
> Any ideas on to how to implement this with ImageJ?
>
> Thanks,
> Cosmin
>  

--
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[Paul Grimm-2]

Hi
That can be done with imageJ
Each cell that is identified as a mask can be identified and added to the ROI manager using the analyze particles function.

run("Analyze Particles...", "size=2000-75000 circularity=0.2-1.00 show=Nothing display exclude clear add");


You can then apply each particle (cell) one at a time as a SELECTION to your other image where the individual vesicles are identified.
something like this

selectImage("Cells");
print(title);

n = roiManager("count");
  for (z=0; z<n; z++) {
      roiManager("select", z);

   run("Copy");
selectImage("Vesicles");
   run("Restore Selection");
run("Analyze Particles...", "minimum=1 maximum=999999 bins=256 show=Nothing display clear");
cells=(nResults);
print(cells);
selectImage(""Cells");
}

This should give you an idea of the macro
--

Paul C. Grimm
Professor of Pediatrics
Dept of Pediatric Nephrology
Lucile Packard Children's Hospital
Stanford University School of Medicine
G306, MC 5208
300 Pasteur Drive
Stanford, CA 94305-5208
USA
phone   [phone]
fax       [phone]


In research, the present devours the past. Sir Peter Medawar

>
>
> Cosmin Mihai wrote:
>> Hi All,
>>
>> I have 2 D images of double stained cells - the cell membrane and some cytoplasmic vesicles – the number of which varies from cell to cell. Each image has 10-30 cells, and both the membrane stain and the vesicle stain can be easily segmented.
>> I am interested to extract a distribution of the vesicle number inside the individual cells at the cell population level, and to do that in an automatic fashion. I was thinking something along the lines of generating a mask from the membrane stain, registering the position and the outline of the cell area, and then apply that information to the image containing the vesicle stain in order to count and assign the number of vesicles to each cell.
>> Any ideas on to how to implement this with ImageJ?
>>
>> Thanks,
>> Cosmin
>>  
>
>
> --
> Rainer Kohler, Ph.D
> Assistant in Research
> Office Phone: [phone]
> Cell Phone: [phone]
> E.mail: [hidden email]
>
> Center for Systems Biology
> Massachusetts General Hospital
> Richard B. Simches Research Center
> 185 Cambridge Street
> Suite 5.210
> Boston, MA 02114 Phone: [phone]
>
>
>
> The information in this e-mail is intended only for the person to whom it is
> addressed. If you believe this e-mail was sent to you in error and the e-mail
> contains patient information, please contact the Partners Compliance HelpLine at
> http://www.partners.org/complianceline . If the e-mail was sent to you in error
> but does not contain patient information, please contact the sender and properly
> dispose of the e-mail.

[yehuda brody]

Hello,
Is there a way to calculate the accurate voxel size (in the Z section) in a wide-field system?

Yehuda Brody
PhD candidate
Institute of Nanotechnology and Advanced Materials,
The Mina & Everard Goodman Faculty of Life Sciences
Bar-Ilan University
Ramat-Gan 52900
Israel


     

[Daniel James White]

Hi Yehuda.

make sure you dont confuse physical sampling voxel spacing with optical resolution.

the optical resolution is limited by diffraction and the N.A.  of the objective lens.
Voxel dimensions are simply whatever you set the hardware up to do for spatial sampling.

If you tell the stage to do 5 micron z steps then thats the z resolution of the images,
but the optical resolution in z is probably nothing like 5 microns.... in this case you might well miss the info in between the z slices.

Anon:
"The point is, the image of a point is not a point"

Its the point spread function, which for a high NA light microscope objective is about 2-3x larger in z than in xy.

you can see what shape it is in 3D by calculating it approximately using Bob Ds ImageJ plugin for that
http://www.optinav.com/Diffraction-PSF-3D.htm

It does not have sharp edges, since its of a size in the order of the wavelength of the light, so that would be impossible.
(Where is the photon exactly?)

But, you can measure the full with at half maximum,
and get some idea of the extent in z direction of the PSF.

This extent is related to the z resolution.

for the xy case, its the famous combination of Abbes equation :
d = lambda / 2N.A.
(there is also an equation for the z case... can you find it?)
and the (arbitrary) Rayleigh criterion for distinguishing 2 point light sources.

Wikipedia will help you out here!

cheers

Dan


Begin forwarded message:

>
> Date:    Sun, 9 May 2010 05:57:49 -0700
> From:    yehuda brody <[hidden email]>
> Subject: Calculating voxel size in wide field system
>
> Hello,
> Is there a way to calculate the accurate voxel size (in the Z section) in a wide-field system?
>
> Yehuda Brody
> PhD candidate
> Institute of Nanotechnology and Advanced Materials,
> The Mina & Everard Goodman Faculty of Life Sciences
> Bar-Ilan University
> Ramat-Gan 52900
> Israel
>

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)

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