Colocalization plugins

[Slavena Trifonova]

Hello,

 

I would ask for some advices for analyzing my data with ImageJ. Shortly what
I did is  immunofluorecent labeling of human Glucocorticoid receptor in
peripheral white blood cells.My aim is to study the temporal localization of
the receptor-(GR shuttling between nucleus and cytoplasm within the time).
Techinically on 10 wells slides I stained white blood cells with Cy3
conjugated Ab  for the Glucocorticoid receptor and Dapi for the nucleus. I
am trying to find an ImageJ plugin that can help me automatically to choose
only the lymphocytes excluding the granulocytes from my pictures and to
measure % of the colocalization of the red and the blue signal . I used
Object counter 3D plug in for segmentation and determination of the cetroids
and intensity centers. After calculation it gives me their respective
coordinates x,y,z.I don’t know how to use these coordinates to calculate the
colocalization I also wonder whether this is the best plugin for the
analysis of my data. I read about this Vector method as well but it is
considered for a method  obtaining a first estimation of colocalization.

Thank you very much for your assistance in advance!

 

Slavena Trifonova

PhD student in Psychoimmunology

Institute of Immunology,

Laboratoire National de Santé

20A rue Auguste Lumière

L-1950

Luxembourg

Grand Duchy of Luxembourg

[phone] *** PLEASE NOTE NEW TELEPHONE NUMBER***

[phone] (fax)

mailto:[hidden email]

http://www.uni-trier.de/ <http://www.uni-trier.de/index.php?id=159>

 

and

 

Laboratory of Immunology,

Centre de Recherche Public - Santé http://www.crp-sante.lu
<http://www.crp-sante.lu/>

 

[John Oreopoulos]

Try the JACoP plugin which allows for thresholding of the images prior to calculating the colocalization. This will only work however if your structures of interest exhibit an intensity that is measurably different than the background signals.

John Oreopoulos


On 2010-07-02, at 9:04 AM, Slavena Trifonova wrote:

> Hello,
>
>
>
> I would ask for some advices for analyzing my data with ImageJ. Shortly what
> I did is  immunofluorecent labeling of human Glucocorticoid receptor in
> peripheral white blood cells.My aim is to study the temporal localization of
> the receptor-(GR shuttling between nucleus and cytoplasm within the time).
> Techinically on 10 wells slides I stained white blood cells with Cy3
> conjugated Ab  for the Glucocorticoid receptor and Dapi for the nucleus. I
> am trying to find an ImageJ plugin that can help me automatically to choose
> only the lymphocytes excluding the granulocytes from my pictures and to
> measure % of the colocalization of the red and the blue signal . I used
> Object counter 3D plug in for segmentation and determination of the cetroids
> and intensity centers. After calculation it gives me their respective
> coordinates x,y,z.I don’t know how to use these coordinates to calculate the
> colocalization I also wonder whether this is the best plugin for the
> analysis of my data. I read about this Vector method as well but it is
> considered for a method  obtaining a first estimation of colocalization.
>
> Thank you very much for your assistance in advance!
>
>
>
> Slavena Trifonova
>
> PhD student in Psychoimmunology
>
> Institute of Immunology,
>
> Laboratoire National de Santé
>
> 20A rue Auguste Lumière
>
> L-1950
>
> Luxembourg
>
> Grand Duchy of Luxembourg
>
> [phone] *** PLEASE NOTE NEW TELEPHONE NUMBER***
>
> [phone] (fax)
>
> mailto:[hidden email]
>
> http://www.uni-trier.de/ <http://www.uni-trier.de/index.php?id=159>
>
>
>
> and
>
>
>
> Laboratory of Immunology,
>
> Centre de Recherche Public - Santé http://www.crp-sante.lu
> <http://www.crp-sante.lu/>
>
>

[Daniel James White]

Dear Slavena,

As John O ponts out the JACoP plugin is nice,
and implements some good methods.

In the Fiji distro of imageJ found at
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
you will find 2 coloc plugins in the analyze menu:
Colocalization threshold - does auto thresholding as per Costes et al, and coloc map and statistics as per Manders et al etc. .
Colocalization test - statistical significance of results - as per Costes

These two plugins are updated and maintained as best we can
(we just fixed a bug in the Rcoloc calculation)

... but at the same time we are already designing and building the next generation
coloc tool for imageJ which we hope will be even more simple to understand and easier to use
than the JACoP plugin.

If you have a wish list for that, just tell us.

For a tutorial on how to use these plugins and what to watch out for
see

http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis

any questions, just ask.

cheers

Dan


On Jul 3, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:

> Date:    Fri, 2 Jul 2010 15:04:02 +0200
> From:    Slavena Trifonova <[hidden email]>
> Subject: Colocalization plugins
>
> Hello,
>
> =20
>
> I would ask for some advices for analyzing my data with ImageJ. Shortly =
> what
> I did is  immunofluorecent labeling of human Glucocorticoid receptor in
> peripheral white blood cells.My aim is to study the temporal =
> localization of
> the receptor-(GR shuttling between nucleus and cytoplasm within the =
> time).
> Techinically on 10 wells slides I stained white blood cells with Cy3
> conjugated Ab  for the Glucocorticoid receptor and Dapi for the nucleus. =
> I
> am trying to find an ImageJ plugin that can help me automatically to =
> choose
> only the lymphocytes excluding the granulocytes from my pictures and to
> measure % of the colocalization of the red and the blue signal . I used
> Object counter 3D plug in for segmentation and determination of the =
> cetroids
> and intensity centers. After calculation it gives me their respective
> coordinates x,y,z.I don=92t know how to use these coordinates to =
> calculate the
> colocalization I also wonder whether this is the best plugin for the
> analysis of my data. I read about this Vector method as well but it is
> considered for a method  obtaining a first estimation of colocalization.
>
> Thank you very much for your assistance in advance!
>
> Slavena Trifonova

> PhD student in Psychoimmunology
> Institute of Immunology,
> Laboratoire National de Sant=E9
> 20A rue Auguste Lumi=E8re
> L-1950
> Luxembourg
> Grand Duchy of Luxembourg
> [phone] *** PLEASE NOTE NEW TELEPHONE NUMBER***
> [phone] (fax)
> mailto:[hidden email]
> http://www.uni-trier.de/ <http://www.uni-trier.de/index.php?id=3D159>=20
>
> and
>
> Laboratory of Immunology
> Centre de Recherche Public - Sant=E9 http://www.crp-sante.lu
> <http://www.crp-sante.lu/>=20
>

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 )

[Slavena Trifonova]

Dear All,

Thank you very much for your replies,I am a beginner in this field. I used
JaCoP manual and tried it on images but still have some questions using this
colocalization measurement plugin. I would really appreciate any help. To
analyze the colocalization of the blue and the red signal which refer to Cy3
for Glucocorticoid receptor and Dapi for nucleus in lymphocytes I focused on
objects based analysis.I choose to work on centre to centre distances
suspecting that the cells are not close to the optical resolution. I don’t
know which picture format I have to use the original file-zvi or it can be a
tif file. I have also some difficulties in setting up the three tabs that
appear on the screen using JaCoP. First I don’t know how to adjust the
threshold of the two images. Second the xy and z (nm) calib -where shall I
take this numbers from and what NA means-it is not pointed out anywhere.
Another issue is how to measure the particle size with imageJ in order to
filter and select only the small lymphocytes excluding the big granulocytes?
Thank you very much for your help in advance.

Kind regards:Slavena Trifonova

> PhD student in Psychoimmunology
>
> Institute of Immunology,
>
> Laboratoire National de Santé
>
> 20A rue Auguste Lumière
>
> L-1950
>
> Luxembourg
>
> Grand Duchy of Luxembourg
>
> [phone] *** PLEASE NOTE NEW TELEPHONE NUMBER***
>
> [phone] (fax)
>
> mailto:[hidden email]
>
> http://www.uni-trier.de/ <http://www.uni-trier.de/index.php?id=159>
>
> and

> Laboratory of Immunology,
>
> Centre de Recherche Public - Santé http://www.crp-sante.lu
> <http://www.crp-sante.lu/>
>

Kind regards:Slavena Trifonova
-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of John
Oreopoulos
Sent: Friday, July 02, 2010 3:42 PM
To: [hidden email]
Subject: Re: Colocalization plugins

Try the JACoP plugin which allows for thresholding of the images prior to
calculating the colocalization. This will only work however if your
structures of interest exhibit an intensity that is measurably different
than the background signals.

John Oreopoulos


On 2010-07-02, at 9:04 AM, Slavena Trifonova wrote:

> Hello,
>
>
>
> I would ask for some advices for analyzing my data with ImageJ. Shortly
what
> I did is  immunofluorecent labeling of human Glucocorticoid receptor in
> peripheral white blood cells.My aim is to study the temporal localization
of
> the receptor-(GR shuttling between nucleus and cytoplasm within the time).
> Techinically on 10 wells slides I stained white blood cells with Cy3
> conjugated Ab  for the Glucocorticoid receptor and Dapi for the nucleus. I
> am trying to find an ImageJ plugin that can help me automatically to
choose
> only the lymphocytes excluding the granulocytes from my pictures and to
> measure % of the colocalization of the red and the blue signal . I used
> Object counter 3D plug in for segmentation and determination of the
cetroids
> and intensity centers. After calculation it gives me their respective
> coordinates x,y,z.I don’t know how to use these coordinates to calculate
the

> colocalization I also wonder whether this is the best plugin for the
> analysis of my data. I read about this Vector method as well but it is
> considered for a method  obtaining a first estimation of colocalization.
>
> Thank you very much for your assistance in advance!
>
>
>
> Slavena Trifonova
>
> PhD student in Psychoimmunology
>
> Institute of Immunology,
>
> Laboratoire National de Santé
>
> 20A rue Auguste Lumière
>
> L-1950
>
> Luxembourg
>
> Grand Duchy of Luxembourg
>
> [phone] *** PLEASE NOTE NEW TELEPHONE NUMBER***
>
> [phone] (fax)
>
> mailto:[hidden email]
>
> http://www.uni-trier.de/ <http://www.uni-trier.de/index.php?id=159>
>
>
>
> and
>
>
>
> Laboratory of Immunology,
>
> Centre de Recherche Public - Santé http://www.crp-sante.lu
> <http://www.crp-sante.lu/>
>
>