Fwd: Question concerning colocalization analysis plugins

[Mina F Abder]

Dear Stéphane, 

I would like to get your input on a colocalisation analysis I am conducting using ICY plugins on confocal images aquired via Spinning disk OBFR microscope.

My biological question is to assess the colocalisation between a protein depicting a nuclear dot pattern (channel 1) and the nucleole which is a specific nuclear region (channel 0) in yeast.

Basically, I want to determine how many dots colocalize with the nucleolus, and if they show total or partial colocalization.

After reading your very interesting paper published recently (Lagache et al, Nat Comm, 2018),  I would like to know whether you recommend me to use your SODA method in my case?

I have attached the ICY protocol I'm using, plus an array of representative images (transmission images before and after Z-stack acquisition, channel 0 and 1 epifluorescent images).

If you could have a look at it and let me know what you think, this would be really great!!

 5325_NO Hoechst_190319_plan-1_1.tif

 5325_NO Hoechst_190319_plan-1_2_C1.tif

 5325_NO Hoechst_190319_plan-1_2_C2.tif

 5325_NO Hoechst_190319_plan-1_3.tif

Also, do not hesistate to ask me for further details.

I thank you very much for allowing me to get your precious feedback.

Sincerely, 

MINA

[Stephane]

Dear Mina,

I'm not sure of what you mean by "partial or complete coloc" but looking at your images it looks like you have only very few objects and i know the colocalization tools bring by SODA (but also the Colocalization Studio you used in your protocol) generally require a minimal number of objects to give accurate results. Thibault (who developed the method) should be able to give more details about it but i'm afraid that your image doesn't contains enough objects for an accurate analysis (i'm not 100% sure but i think so) so i would be better if you can increase the density of objects or work on larger image. Also it's very important to correctly define the "cell mask" which define the area where we are looking for colocalization as obviously results can be very different if we consider the whole image or only where we have cells ;)

Still i suggest you to use the Soda Suite block :

This block should provide you the best analysis / results for objects colocalization.

Hope this help :)

Best,

- Stephane

[Thibault Lagache]

Hi Mina,

Here Thibault, the developer of SODA method. Your problem is not that simple and I am not sure that SODA would apply due to the few number of objects around each nucleolus (I can't really see any colocalization, it seems to me that objects are close to the nucleolus but not inside).

What you could do is measuring the distance of objects to the nucleolus and test whether this proximity is due to chance (but for this you would need to localize the nucleus membrane) and also look if this distance changes between conditions.

Let me know if I was clear and if we could further help,

Thanks

Thibault

[Mina F Abder]

Dear Stephane and Thibault,

I thank warmly both of you for your valuable inputs !

I've had interesting brainstorming sessions with my thesis supervisors during this week.

Indeed, SODA method is not really suitable for our analysis.

We are going to follow your pieces of avice and analyze the geometric distribution of the dots around the nucleolus.

Thanks again !

Sincerly,

MINA

--
You received this message because you are subscribed to the Google Groups "Icy imaging" group.
To unsubscribe from this group and stop receiving emails from it, send an email to icy-software...@googlegroups.com.
To post to this group, send email to icy-so...@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msgid/icy-software/e8c0d5b0-b37f-4172-9577-c82e3cdad481%40googlegroups.com.
For more options, visit https://groups.google.com/d/optout.