Posted by
Daniel James White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Dyes-for-apoplastic-pH-in-leaf-tissue-tp5505884p5511006.html
Hi Leong,
On Sep 8, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:
>
> Date: Tue, 7 Sep 2010 13:59:37 -0500
> From: Teng-Leong Chew <
[hidden email]>
> Subject: Colocalization question
>
> Hi all,
>
> Pardon me for opening up a can of worms of colocalization issue.
>
no need to excuse yourself for asking a sensible question!
> I wonder if anyone has experience in thresholding a biological
> object using a channel (say, green actin in a contractile ring)
> and use only that selected ROI to quantify Pearson coeefficient
> between green and a red-labeled protein? Bottom line question
> is: how much of the red protein co-localizes to green within
> the object and not the whole cell?
Its a pretty sensible idea to do something like that.
Rarely should one analyze the whole of an image,
as the biology one is interested in is only contained in a certain region of interest.
Thus one should only analyse that region of interest.
So, you need a good way to make the region of interest.
One way is just to draw it (a bit subjective)
but another more objective way, as you suggest is to use a simple threshold or other clever way to segment one of the channels,
and use the result as the mask to do the colocalization measurement in.
I suggest using the Costes methods for auto thresholding (in addition to the ROI or Mask)
and statistical significance
and to report both Pearsons coefficient for the masked/RIO area and Pearsons above the auto threshold levels,
and the auto thresholds,
and the Manders coefficients M1 and M2 and thresholded Manders tM1 and tM2
Manders coeff tell you about coloc from the perspective of each channel,
and thats what you mentioned you want to know i believe.
Lots of details and pitfalls are explained at
http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis>
> We have Volocity, MetaMorph and ImageJ.
>
Fiji is just imageJ, but has the latest bug fixes
(by us Fiji-ers - including a bug spotted by the Volocity guys which eas recenbtly fixed)
of the ImageJ plugins:
Colocalization Threshold and Test
along with lots of other nice stuff added on top of imageJ.
(Fiji is becoming the de facto ImageJ distro of choice for cell biology etc.
and you wont find the latest bug fixes for colo in the other plugin distributions I expect.)
I don't know about metamorph coloc analysis - never saw it
but Volocity is very good and has been apparently improved recently on coloc analysis....
(but its not open source so you cant check the maths yourself unfortunately... its a very nice black box.)
> Thanks in advance for your help.
I'm always happy to help with Colocalization!!!
cheers
Dan
>
> Regards,
> Leong
>
>
> --
> Teng-Leong Chew, Ph.D.
> Director, Cell Imaging Facility & Nikon Imaging Center
> Director of University Imaging Resources
> Feinberg School of Medicine & Office of Research
> Northwestern University
> 303 E. Chicago Avenue
> Chicago, IL 60611
> (312) 503-2841
> (847) 491-7091 (W, F)
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 )