Colocalization over 3 channels

[Magali SOYER]

Hi,

I'm trying to analyse colocaliZation of 3 endosomal markers. I've tried the Colocalization Studio, it works fine to compare 2 channels at a time, but do you think I could do it for 3 channels?
I've also tried the "Colocalizer" protocol that Fabrice wrote, do you think it would be easier to add a layer here or would that be too complicated?


What I have in mind -and tried for 2 channels at a time:
-1/ outline the cells: Z-proj in red channel + active contours
-2/ Use Spot Detector separately in the 3 channels to detect the vesicules
-3/ Use the Colocalization Studio or the Colocalizer protocol to analyse the colocalisation
-4/ Get an Excel file with a summary for each cell.

Do you think we can do something like that?

I can't attach a file here as the stacks are apparently too big...can try to send it to whoever wants to/can help (Fabrice, Thibault?? still have your emails somewhere I think!)

Regards,

Magali

[thibault...@yahoo.fr]

Hi Magali,

I think the best should be to use the colocalizationstudio_object block in a protocol; indeed, if you use it a first time for channels 1 and 2, then you can get the spots 2 that colocalize with spots 1 as an output of the block. You can then take these "colocalized spots 2" to test whether or not they colocalize with spots 3 from the channel 3. You can then get the trouples "spots 3 that colocalize with spots 2 that colocalize with spots 1". Could this answer your point?

If not, we can discuss and I could develop something specific for you ; )

Best regards,

Thibault

[Magali SOYER]

Hi Thibault,

Sorry to get back to you so late, I had to check with the former post-doc whose work I'm finishing if the "spots 3 that colocalise with spots 2 that colocalise with spots 1" option was okay for her.
I'll try to built the protocol this week; hopefully I can manage to do it myself but you know how bad I am with that computing part so you might get another email soon :)

Best regards,

Mag 

[thibault...@yahoo.fr]

Hi Magali, No problem, let me know if you have any trouble building your protocol!

Thibault

[Alessandro Di Maio]

Hello Thibault,

I'm relatively new to ICY as I started to play with it after Alexander's presentation in Cambridge (early Dec). Great!

I have a similar question as Magali and I've been trying to build a custom protocol in order to get reliable data out of a multiple colocalization analysis. Here are few details:

I need to see weather

- Ch0 coloc. with Ch1

- Ch1 coloc. with Ch2

- Ch0 coloc. with Ch2

- Ch1 colocalizing Ch2 (Ch1+Ch2) coloc with Ch3

- Ch0+Ch1 coloc with Ch3

- Ch0+Ch2 coloc with Ch3

So far I've been able to use the colocalizer protocol to get the first three combinations but I did not have much luck to have the colocalized components as output. As I have a lot of images, I was hoping to get a reasonably fast analysis and using protocols I feel I'm close but at this point I'm lost.

Any help would be greatly appreciated. Merci.

Congrats for the amazing software.

All my Best!

Alex

[Thibault Lagache]

Dear Alex,

Thanks for your interest and sorry for the late answer. Attached, a test protocol with colocalization between: Ch1 with 2, Ch 1 with 3, Ch 2 with 3 and Channel (1 with 2 ) with 3. Then, you can adapt it to your needs, especially, I didn't put the workbook to file blocks.

Best regards

Thibault

[Alessandro Di Maio]

Hello Thibault,

thank you so much for the info. Much appreciated! Congrats for the software! the more I use it the more i like it!!

I've been actually playing with the protocols and I created a (quite complicated) colocalization protocol and I would greatly appreciate if you could give me any feedback on it (attached).

I did run few samples and it seems to work (it does not crush....) but I'm still in the process of validating my results with a classic (extremely slow) manual counting.

Particularly I did base the analysis on the basic colocalization protocol adding few plug-ins.

If you think it's reliable and doesn't have any gap, I'd like to use to run multiple files (batch). How would I do so?

thanks a lot in advance for you help! Greatly Appreciated!!

All my Best,

Alex

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[Thibault Lagache]

Dear Alex

I didn't understand that you used the colocalizer block. You should use the colocalization Studio Object block which is more robust (this is the block I used in the protocol I attached before)

To validate your protocol you can generate images with known level of colocalization (plugin Colocalization Simulator).

I do not see "strange" things in your protocol.

All the best

Thibault

[cga....@gmail.com]

Dear Thibault,

Thanks for posting the protocol for 3 color colocalization.

I am trying to analyse colocalization in a ROI. So I added the suggested ROI from sequence. The protocol runs fine until the last block 16. There it generates an error.

Is it because there is no info on where to export to in excell, like the block EXport singles that I saw in SODA 2 protocol?

I would also like to export single spots, per ROI. How can I do it?

Thanks

PS I have attached the protocol I am trying to use.

[Thibault Lagache]

Hi,

What is the error message?

Thanks

Thibault

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<prtocol_3ChannelsROI.protocol>

[Cláudia Almeida]

Hi

There was no error message but block 16 never got GREEN.

thanks

Claudia

Thibault

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