http://confocal-microscopy-list.275.s1.nabble.com/Colocalization-on-Z-stack-images-tp7580319p7580349.html
Thank you so much for your kind reply and valuable information. I'd
analysis. It's the first time that I'm using it, it seems good but I
> Dear Marjan,
>
> apologies for the delay, I have been away on annual leave.
> It is hard to tell from your description what might work best, since I
> don't know what the structures are. In any case, you need something
> that gives you good values in 3D. It is tempting to flatten the
> z-stack, then do a Pearson's or something similar, but that is bad
> practice, as you are ignoring the third dimension (z). Look which ones
> do proper 3D. coloc2 (in Fiji), is a good one. The object-based Moore
> plugin (
http://crg.ubc.ca/moore/) looks also good, although I haven't
> tried it myself. If the structures you're looking at are round(ish),
> then I must say that I have been very happy with the algorithm we have
> produced. This will also give you a clear picture depending on the
> z-level (e.g. if the structures colocalise more outside or inside the
> nucleus) and the distances between the structures. I don't have the
> paper here, but can send it to you tomorrow; the plugin requires
> Matlab. Maybe, if it strongly depends on the z-level, it may be a
> good-enough start to look at single planes from different z-levels
> (using a 2D approach, e.g. Manders or Pearsons) and show that they are
> very different.
> Colocalisation is a tricky subject - I've ended up spending a lot more
> time on it than I ever thought I would.
>
> Let me know if this helps; if not, just get back to me, I'm now back at
> work.
>
> With kind regards
>
> Philippe
>
> On Thu, May 9, 2013 at 1:24 PM, Marjan Gharagozloo <
[hidden email]>
> wrote:
>> Dear Philippe,
>>
>> Thank you so much for your reply. I'm looking for RNA translocation
>> inside nucleus. The RNA has been labeled with Cy3 and Nuclei are
>> stained with Draq5. This is my first confocal experience and I'm
>> really confused! My problem is that the quantity of colocalization is
>> changing by different focal plane. So, I tried looking at Z-stacks to
>> find something reliable. I used Zeiss software for Colocalization (LSM
>> 710), I got some data but I'm not happy with them because I'm not sure
>> if they are showing the correct amount of colocalization. Then, I
>> checked imageJ, actually I found Many ImageJ software online
>> (Imagej2X, WCIF ImageJ, and just ImageJ). Also I downloaded Huygens
>> and Volocity 3D, but I couldn't find something related to my need.
>>
>> Could you kindly tell me which one is more relevant to my case? Any
>> suggestion would be highly appreciated.
>>
>>
>> Bests
>> Marjan
>>
>>
>> On 5/8/13, phil laissue <
[hidden email]> wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>> *****
>>>
>>> Hi Marjan,
>>>
>>> more information would be needed to understand the problem, but I'm
>>> pasting in a few references below. If you have a z-stack, the best
>>> option is to do a 3D colocalisation analysis by including all focal
>>> planes (hopefully you acquired using Nyquist-Shannon reconstruction
>>> theorem).
>>>
>>> (from an earlier post):
>>> By no means a comprehensive list, but in my humble opinion some of the
>>> most user-friendly discussions/approaches. Really depends on the
>>> structures in question, there's not one single approach that works
>>> best.
>>>
>>> pixel-based:
>>>
http://www.ncbi.nlm.nih.gov/pubmed/23026999>>> diva-portal.org/smash/get/diva2:563664/FULLTEXT02
>>> coloc2:
>>>
http://fiji.sc/Colocalization_Analysis>>>
http://www.ncbi.nlm.nih.gov/pubmed/21209361>>>
http://www.ncbi.nlm.nih.gov/pubmed/15189895>>> object-based:
>>>
http://www.ncbi.nlm.nih.gov/pubmed/20858446>>>
http://crg.ubc.ca/moore/>>>
http://www.ncbi.nlm.nih.gov/pubmed/19746416>>>
>>>
http://www.ncbi.nlm.nih.gov/pubmed/23381680>>> (happy to send you reprint and matlab code)
>>>
>>> Also worth checking out:
>>>
http://www.ncbi.nlm.nih.gov/pubmed/17210054>>>
http://www.ncbi.nlm.nih.gov/pubmed/22086768>>>
>>> Hope this helps. Kind regards
>>>
>>> Philippe
>>>
>>> ______________________________
>>> Philippe Laissue, PhD, Bioimaging Manager
>>> School of Biological Sciences, Room 4.17
>>> University of Essex, Colchester CO4 3SQ, UK
>>> (0044) 01206 872246 / (0044) 07842 676 456
>>>
[hidden email]
>>> privatewww.essex.ac.uk/~plaissue
>>>
>>> On Wed, May 8, 2013 at 5:44 PM, Marjan Gharagozloo <
[hidden email]>
>>> wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>>>> *****
>>>>
>>>> Dear All,
>>>>
>>>> I'd like to study colocalization of Draq5 and Cy3 in Nucleus.
>>>> Actually, this topic is totally new to me and I've read many articles
>>>> and
>>>> manuals to find a way for quantifying colocalization. However, I don't
>>>> know how I can analyze colocalization in my Zstack images. I know by
>>>> changing focal plane, I'll get different results and quantities. It's
>>>> kind of you if send me some information to do this analysis.
>>>>
>>>> Many thanks and best regards:
>>>> Marjan
>>>>
>>>> --
>>>> Best regards:
>>>>
>>>> Marjan Gharagozloo (PhD)
>>>> Postdoctoral Fellow
>>>> University of Waterloo
>>>> School of Pharmacy, PHR3002
>>>> 10 Victoria St S, N2G 2B2
>>>> Kitchener, ON, Canada
>