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Re: Automatic Cell Counting

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Barron, Francie

Re: Automatic Cell Counting

Hi,

I'm new to this program, so hope to receive some help.

I have paraffin sections through a mouse E11.5 mandibular arch that I have stained for proliferation and cell death. I wanted to count the total number of cells (via DAPI) and compare that to cell proliferation positive and cell death positive cells in both control and mutant (I have them stored as 16-bit RGB as well as 8-bit). As of now, I've been using the manual cell counter plug-in through Fuji, but would like a way to specify an ROI and automate this process. I have downloaded and tried the UCSB ITCN plugin for automation, but it was not able to count cells closely packed together despite a low threshold. If anyone can suggest a way to automate this (a new plugin or one contained into ImageJ), or help with the plugins that I have that would be great.

Thanks so much!!
~Francie

NOTE NEW EMAIL ADDRESS: [hidden email]

***************************************************************

Francie Barron (Hyndman), Graduate Student
Cells Biology, Stem Cells, and Development Program
Dr. David Clouthier lab, Craniofacial Biology

For Correspondence:
University of Colorado, Health Sciences Center
Department of Craniofacial Biology
Mailstop 8120
P.O. Box 6511
Aurora, CO 80045

For Deliveries:
University of Colorado Denver Health Sciences Center
Department of Craniofacial Biology
Building RC1 South, Room 11400B
12801 East 17th Avenue
Aurora, Co 80010

Lab: 303-724-4566
Fax: 303-724-4580

*****************************************************************

Daniel James White

Re: Automatic Cell Counting

Hi Francie,

send me a sample image and i will make some suggestions....touching nuclei are often very hard to get.

A watershed method might work, see watershed here.
http://rsbweb.nih.gov/ij/docs/menus/process.html#binary

cheers

Dan

On Jul 2, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:

>
> Date: Wed, 30 Jun 2010 22:51:15 -0600
> From: "Barron, Francie" <[hidden email]>
> Subject: Re: Automatic Cell Counting
>
> Hi,
>
> I'm new to this program, so hope to receive some help.
>
> I have paraffin sections through a mouse E11.5 mandibular arch that I have =
> stained for proliferation and cell death. I wanted to count the total numb=
> er of cells (via DAPI) and compare that to cell proliferation positive and =
> cell death positive cells in both control and mutant (I have them stored as=
> 16-bit RGB as well as 8-bit). As of now, I've been using the manual cell =
> counter plug-in through Fuji, but would like a way to specify an ROI and au=
> tomate this process. I have downloaded and tried the UCSB ITCN plugin for =
> automation, but it was not able to count cells closely packed together desp=
> ite a low threshold. If anyone can suggest a way to automate this (a new p=
> lugin or one contained into ImageJ), or help with the plugins that I have t=
> hat would be great.
>
> Thanks so much!!
> ~Francie
>

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Processing and Analysis
Light Microscopy Facility
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

New Mobile Number!!!

+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
+49 (0)351 210 1078 (Fax MPI-CBG LMF)

http://www.bioimagexd.net
http://www.chalkie.org.uk
[hidden email]
( [hidden email] )

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

+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
+49 (0)351 210 1078 (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 )

Barron, Francie

Re: Automatic Cell Counting

Hi Dan,

What is the email I can send the information to?

Thanks,
~Francie

NOTE NEW EMAIL ADDRESS: [hidden email]

***************************************************************

Francie Barron (Hyndman), Graduate Student
Cells Biology, Stem Cells, and Development Program
Dr. David Clouthier lab, Craniofacial Biology

For Correspondence:
University of Colorado, Health Sciences Center
Department of Craniofacial Biology
Mailstop 8120
P.O. Box 6511
Aurora, CO 80045

For Deliveries:
University of Colorado Denver Health Sciences Center
Department of Craniofacial Biology
Building RC1 South, Room 11400B
12801 East 17th Avenue
Aurora, Co 80010

Lab: 303-724-4566
Fax: 303-724-4580

*****************************************************************

________________________________________
From: ImageJ Interest Group [[hidden email]] On Behalf Of Daniel James White [[hidden email]]
Sent: Friday, July 02, 2010 3:50 AM
To: [hidden email]
Subject: Re: Automatic Cell Counting

Hi Francie,

send me a sample image and i will make some suggestions....touching nuclei are often very hard to get.

A watershed method might work, see watershed here.
http://rsbweb.nih.gov/ij/docs/menus/process.html#binary

cheers

Dan

On Jul 2, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:

Daniel James White

Re: Automatic Cell Counting

In reply to this post by Barron, Francie

Hi Francie,

these images are very nice....
but there is little chance that any simple method on the computer will be able to correctly segment
most of that DAPI nuclei in that image.

Even the other red channel is hard.

the objects overlap too much and there is no intensity max at their centre you could use.
THer tissue is very densly packed.

In this case you need something quite high tech,
where you can use a pipeline of methods to get the nuclei you want.

CellProfiler might be a good palce to start,
ir a macro or script in imageJ/Fiji

you need to find a local collaborator to help you out.

If you want to kist know the average brightness of the nuclei, that is possible my estimating the number
and measuring a few cells, then measuring the total intensity in the image.

what is it that you actually want to know from these images?

cheers

Dan

On Jul 7, 2010, at 7:17 PM, Barron, Francie wrote:

> Hi Dan,
>
> I converted to jpg, cut it in half on size. Hopefully this goes though.
>
> ~Francie
>
> NOTE NEW EMAIL ADDRESS: [hidden email]
>
> ***************************************************************
>
> Francie Barron (Hyndman), Graduate Student
> Cells Biology, Stem Cells, and Development Program
> Dr. David Clouthier lab, Craniofacial Biology
>
> For Correspondence:
> University of Colorado, Health Sciences Center
> Department of Craniofacial Biology
> Mailstop 8120
> P.O. Box 6511
> Aurora, CO 80045
>
> For Deliveries:
> University of Colorado Denver Health Sciences Center
> Department of Craniofacial Biology
> Building RC1 South, Room 11400B
> 12801 East 17th Avenue
> Aurora, Co 80010
>
> Lab: 303-724-4566
> Fax: 303-724-4580
>
> *****************************************************************
>
> ________________________________________
> From: Daniel James White [[hidden email]]
> Sent: Wednesday, July 07, 2010 9:18 AM
> To: Barron, Francie
> Subject: Re:Automatic Cell Counting
>
> to me!
>
> [hidden email]
>
> just reply to this emial
>
> Dan
>
>
> On Jul 7, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:
>
>> Date: Tue, 6 Jul 2010 10:07:46 -0600
>> From: "Barron, Francie" <[hidden email]>
>> Subject: Re: Automatic Cell Counting
>>
>> Hi Dan,
>>
>> What is the email I can send the information to?
>>
>> Thanks,
>> ~Francie
>>
>> NOTE NEW EMAIL ADDRESS: [hidden email]
>
> 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
>
> +49 (0)15114966933 (German Mobile)
> +49 (0)351 210 2627 (Work phone at MPI-CBG)
> +49 (0)351 210 1078 (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 )
>
>
>
>
>
>
>
>
>
>
>
> <DAPImontage.jpg><REDmontage.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

+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
+49 (0)351 210 1078 (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 )