Colocalization with Coloc2

[Knecht, David]

I was trying to use the Coloc2 in Fiji and the colocsample data provided in the cookbook. (https://imagej.net/Colocalization_Analysis) to instruct my microscopy students.  I have not done much of this myself so wanted to understand the software before teaching it.

I was unable to generate any useful data.   I got all kinds of warnings when I ran it with the stacks or a single slice from the stacks even though the dataset was clearly colocalized.

1.   I tried to use a freehand ROI to focus on the membrane edge of the cell, but it did not seem to be used or make any difference.  The image shown in the results was always the entire image and nothing seemed to indictate it was working with just the ROI
2.  I tried subtracting background (around 60) from the images so only real data was analyzed and still got lots of warnings.
3.  The results scatterplots seemed meaningless. In no case did it show the high degree of colocalization of the two probes in the sample data.
4.   I am not sure how to generate the useful dot scatterplots of the sort shown in Dunn et al. from this analysis.
5.  With the stacks, I expected a slice by slice analysis.  The image shown in the results seemed to be only the first slice.
6.  I was never able to see an image with the colocalized pixels found by the analysis highlighted.

It would be really useful to someone doing this for the first time if that page were updated with a complete astep by step nalysis of that sample data.  Settings, results, etc. with different inputs (ROI, noise reduction, stacks vs. slices etc.). so all the complexities are clarified.  Is that available somewhere?   Thanks- Dave

Dr. David Knecht
Professor, Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125
860-486-2200


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[CARL Philippe (LBP)]

Dear David,
I would rather recommend you to use the Colocalization Finder plugin:
https://imagejdocu.tudor.lu/plugin/analysis/colocalizationfinder/start
for which I took over the maintenance.
The plugin can be applied for whatever bit depth picture (i.e. 8, 16 or 32 Bit picture) and able you to define "on the fly" an analysis ROI (i.e. within the scatterPlot picture) as well as within the composite picture.
The calculations are generated (inside a results window) when you click inside a ROI (i.e. within the scatterPlot or composite picture) and you can as well set a ROI selection with a double click inside a ROI.
Nevertheless, the plugin has only be written for the analysis of two single pictures as start conditions (i.e. not for a stack).
But the code is very easily scriptable for stacks and you can find two example macros within the website link I indicted higher.
The only drawback of the tool is maybe the lack of a good "biologist compatible" description (sorry I'm only a physicist) of it's features for which I'm waiting for some "biologist colleagues" to write it and you are as well welcome to do so if you wish.
Feel free to contact me if you have any issues with this tool or ideas for improving it.
My best regards,
Philippe

Philippe CARL
Laboratoire de Bioimagerie et Pathologies
UMR 7021 CNRS - Université de Strasbourg
Faculté de Pharmacie
74 route du Rhin
67401 ILLKIRCH
Tel : [phone]

----- Mail original -----
De: "Knecht, David" <[hidden email]>
À: "imagej" <[hidden email]>
Envoyé: Vendredi 6 Décembre 2019 16:56:21
Objet: Colocalization with Coloc2

I was trying to use the Coloc2 in Fiji and the colocsample data provided in the cookbook. (https://imagej.net/Colocalization_Analysis) to instruct my microscopy students.  I have not done much of this myself so wanted to understand the software before teaching it.

I was unable to generate any useful data.   I got all kinds of warnings when I ran it with the stacks or a single slice from the stacks even though the dataset was clearly colocalized.

1.   I tried to use a freehand ROI to focus on the membrane edge of the cell, but it did not seem to be used or make any difference.  The image shown in the results was always the entire image and nothing seemed to indictate it was working with just the ROI
2.  I tried subtracting background (around 60) from the images so only real data was analyzed and still got lots of warnings.
3.  The results scatterplots seemed meaningless. In no case did it show the high degree of colocalization of the two probes in the sample data.
4.   I am not sure how to generate the useful dot scatterplots of the sort shown in Dunn et al. from this analysis.
5.  With the stacks, I expected a slice by slice analysis.  The image shown in the results seemed to be only the first slice.
6.  I was never able to see an image with the colocalized pixels found by the analysis highlighted.

It would be really useful to someone doing this for the first time if that page were updated with a complete astep by step nalysis of that sample data.  Settings, results, etc. with different inputs (ROI, noise reduction, stacks vs. slices etc.). so all the complexities are clarified.  Is that available somewhere?   Thanks- Dave

Dr. David Knecht
Professor, Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125
860-486-2200


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[Ellen Arena-2]

David…

Ah the joys of colocalization analysis!  It’s a pain - I know.  And I understand the frustration with using Coloc2… it was made to point-out/highlight the pitfalls in colocalization analyses - of which there are many - hence, all the warning messages.  If I were you - I’d just start by reading through these two pages on the ImageJ wiki:

https://imagej.net/Colocalization_Analysis <https://imagej.net/Colocalization_Analysis>
https://imagej.net/Coloc_2 <https://imagej.net/Coloc_2>

They’ll provide more info on Colocalization in general  - as well as more details on the plugin itself.  When was the last time you updated your Fiji installation?  There should be no issue with running a z-stack (a max projection should be displayed instead of only the first slice - that has been updated - but the analysis is on the whole stack)… there were somewhat recent updates made (https://forum.image.sc/t/release-of-coloc-2-version-3-0-0/5099 <https://forum.image.sc/t/release-of-coloc-2-version-3-0-0/5099>) - so just try updating things and running it again.  For the ROI issue… you can try using a mask instead - selecting it in the dropdown menu…  if this still doesn’t work - let me know.

You can also check out another colocalization plugin, JACoP, which also addresses object-based overlap analysis.

https://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:jacop_2.0:just_another_colocalization_plugin:start <https://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:jacop_2.0:just_another_colocalization_plugin:start>

Too - new methods were recently developed by colleagues, Shulei Wang and Ming Yuan, RKColocal: http://www.columbia.edu/~my2550/rkcolocal-Introduction.html <http://www.columbia.edu/~my2550/rkcolocal-Introduction.html>  This code is all available to run… in R.  You will be able to get those colocalized pixels you are looking for using this new technique.  I am currently moving that code over into the IJ2 framework (https://github.com/imagej/imagej-ops/commit/7870ad17a3cd36e5c9915580a25d53da5893f5c3 <https://github.com/imagej/imagej-ops/commit/7870ad17a3cd36e5c9915580a25d53da5893f5c3>), but only the global method is currently available (hopefully, the pixel wise will be coming soon!).

If you have other specific questions - just let us/me know!  I also frequent the Scientific Community Image Forum (https://forum.image.sc/ <https://forum.image.sc/>) - and there are tons of posts there regarding Coloc2 that you might find helpful (https://forum.image.sc/tags/coloc2 <https://forum.image.sc/tags/coloc2>).

eta

> On Dec 6, 2019, at 11:08 AM, CARL Philippe (LBP) <[hidden email]> wrote:
>
> Dear David,
> I would rather recommend you to use the Colocalization Finder plugin:
> https://imagejdocu.tudor.lu/plugin/analysis/colocalizationfinder/start
> for which I took over the maintenance.
> The plugin can be applied for whatever bit depth picture (i.e. 8, 16 or 32 Bit picture) and able you to define "on the fly" an analysis ROI (i.e. within the scatterPlot picture) as well as within the composite picture.
> The calculations are generated (inside a results window) when you click inside a ROI (i.e. within the scatterPlot or composite picture) and you can as well set a ROI selection with a double click inside a ROI.
> Nevertheless, the plugin has only be written for the analysis of two single pictures as start conditions (i.e. not for a stack).
> But the code is very easily scriptable for stacks and you can find two example macros within the website link I indicted higher.
> The only drawback of the tool is maybe the lack of a good "biologist compatible" description (sorry I'm only a physicist) of it's features for which I'm waiting for some "biologist colleagues" to write it and you are as well welcome to do so if you wish.
> Feel free to contact me if you have any issues with this tool or ideas for improving it.
> My best regards,
> Philippe
>
> Philippe CARL
> Laboratoire de Bioimagerie et Pathologies
> UMR 7021 CNRS - Université de Strasbourg
> Faculté de Pharmacie
> 74 route du Rhin
> 67401 ILLKIRCH
> Tel : [phone]
>
> ----- Mail original -----
> De: "Knecht, David" <[hidden email]>
> À: "imagej" <[hidden email]>
> Envoyé: Vendredi 6 Décembre 2019 16:56:21
> Objet: Colocalization with Coloc2
>
> I was trying to use the Coloc2 in Fiji and the colocsample data provided in the cookbook. (https://imagej.net/Colocalization_Analysis) to instruct my microscopy students.  I have not done much of this myself so wanted to understand the software before teaching it.
>
> I was unable to generate any useful data.   I got all kinds of warnings when I ran it with the stacks or a single slice from the stacks even though the dataset was clearly colocalized.
>
> 1.   I tried to use a freehand ROI to focus on the membrane edge of the cell, but it did not seem to be used or make any difference.  The image shown in the results was always the entire image and nothing seemed to indictate it was working with just the ROI
> 2.  I tried subtracting background (around 60) from the images so only real data was analyzed and still got lots of warnings.
> 3.  The results scatterplots seemed meaningless. In no case did it show the high degree of colocalization of the two probes in the sample data.
> 4.   I am not sure how to generate the useful dot scatterplots of the sort shown in Dunn et al. from this analysis.
> 5.  With the stacks, I expected a slice by slice analysis.  The image shown in the results seemed to be only the first slice.
> 6.  I was never able to see an image with the colocalized pixels found by the analysis highlighted.
>
> It would be really useful to someone doing this for the first time if that page were updated with a complete astep by step nalysis of that sample data.  Settings, results, etc. with different inputs (ROI, noise reduction, stacks vs. slices etc.). so all the complexities are clarified.  Is that available somewhere?   Thanks- Dave
>
> Dr. David Knecht
> Professor, Department of Molecular and Cell Biology
> University of Connecticut
> 91 N. Eagleville Rd.
> U-3125
> Storrs, CT 06269-3125
> 860-486-2200
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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