Re: Quantifying colocalization and measuring focal adhesion colocalization in puncta
There are many different tools for analysing colocalisation and I can't really recommend one specific one as it very much depends on the precise nature of your images and analysis task. However, NEUBIAS academy has just run an 'Introduction to Colocalization' webinar this week. You can find a recording of the webinar here: http://eubias.org/NEUBIAS/training-schools/neubias-academy-home/. This will be followed up next week by a webinar on 'Advanced colocalization methods for SMLM and statistical tools for analyzing the spatial distribution of objects'. So, if you are new to colocalisation analysis it might be worth having a look at these.
Quite obviously you have applications which are quite similar to some we are doing within our laboratory.
The way we are performing this is to isolate the FA through an Image>Adjust>Threshold... and identify them with an Analyze>Analyze_Particles...
Then we use the following plugin for measuring the colocalization:
http://questpharma.u-strasbg.fr/html/colocalization-finder.html There are several colocalization plugins out there, but up to my knowledge this is the only tool able to define an analysis ROI on the fly.
At least it is for performing the analysis like the one I describe here that I implemented this feature to the plugin.
Following the Analyze_Particles you can make a More>>OR_(Combine) in order to measure the colocalization of all the FA in one shot.
But given that the plugin is (as well on the fly) macro scriptable, I would rather analyze each FA ROI idependantly (within a loop) so that you can extract way more information from the obtained results (like look for the colocalication results of a given FA as a function of it's area, aspect ration,...).
Also in the case you have critics about the plugin or ideas on how to improve it (or even it's description within the website) do not hesitate to contact me.
My best regards,
Laboratoire de Bioimagerie et Pathologies
UMR 7021 CNRS - Université de Strasbourg
Faculté de Pharmacie
74 route du Rhin
Tel : +33(0)3 68 85 42 89
----- Mail original -----
De: "Grace Ciabattoni" <[hidden email]>
Envoyé: Samedi 3 Octobre 2020 05:12:09
Objet: Quantifying colocalization and measuring focal adhesion colocalization in puncta
What is the best way to measure the changes in focal adhesion colocalization? Do you measure the individual puncta, or whole cell fluorescence?
Additionally, does anyone have any recommendations for quantifying the amount of colocalization in immunofluorescence?