Colocalization within Regions of Interest

[Oleg Broytman]

Greetings.  I need some advice about how to use Image J to carry out my colocalization analysis.  I am studying changes in protein localization in the Phrenic Motor Nucleus (PMN)- a cluster of neurons in the cervical spinal cord.  I have acquired Z-stack images under the confocal microscope of PMN neurons.  The PMN neurons are immunostained for Protein 1 on the Green channel of fluorescence and Protein 2 on the Red Channel of fluorescence.  They are also labeled with a tracer dye, the fluorescence of which is acquired on the Far Red channel.

What I would like to do is to analyze the extent of colocalization between Protein 1 and Protein 2 - not in the entire field, but only in the regions positively stained with the tracer dye.  So, I'd like to select regions of interest in each slice of the Z-stack in the Far Red channel (which would generate a whole list of ROIs for each stack), and then analyze Green/Red colocalization within each region of interest.

I could really use some advice on how to do this.  All the colocalization plugins I've encountered thus far analyze 2D colocalization in the entire 2D image, or 3D colocalization for the entire stack.  Is there a way to do colocalization only within a specific region of interest?

Thank you in advance for your help.

Sincerely,

Oleg Broytman
University of Wisconsin - Madison.

[John Oreopoulos]

Dear Oleg,

I highly recommend reading a very recent review on colocalization by the Dunn group that discusses this very aspect (about limiting the analysis to ROIs and when this is or is not appropriate):

Dunn, K.W., M.M. Kamocka, and J.H. McDonald, A practical guide to evaluating colocalization in biological microscopy. American Journal of Physiology-Cell Physiology, 2011. 300(4): p. C723-C742.

As I recall, I think the authors even listed some different ways to implement ROI colocalization analysis with commercial and free colocalization software that are already widely available. For example, Costes' automatic threshold approach (which is available in the JACoP plugin) is discussed at length.

Cheers,

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2011-05-18, at 8:44 PM, Oleg Broytman wrote:

> Greetings.  I need some advice about how to use Image J to carry out my colocalization analysis.  I am studying changes in protein localization in the Phrenic Motor Nucleus (PMN)- a cluster of neurons in the cervical spinal cord.  I have acquired Z-stack images under the confocal microscope of PMN neurons.  The PMN neurons are immunostained for Protein 1 on the Green channel of fluorescence and Protein 2 on the Red Channel of fluorescence.  They are also labeled with a tracer dye, the fluorescence of which is acquired on the Far Red channel.
>
> What I would like to do is to analyze the extent of colocalization between Protein 1 and Protein 2 - not in the entire field, but only in the regions positively stained with the tracer dye.  So, I'd like to select regions of interest in each slice of the Z-stack in the Far Red channel (which would generate a whole list of ROIs for each stack), and then analyze Green/Red colocalization within each region of interest.
>
> I could really use some advice on how to do this.  All the colocalization plugins I've encountered thus far analyze 2D colocalization in the entire 2D image, or 3D colocalization for the entire stack.  Is there a way to do colocalization only within a specific region of interest?
>
> Thank you in advance for your help.
>
> Sincerely,
>
> Oleg Broytman
> University of Wisconsin - Madison.

[srai]

Hello Oleg

John's  suggestion must have probably already helped you.

What I usually do for looking at colocalisation at a certain region of interest is to select the region and then duplicate the region for the whole z stack. Then perform the colocalisation tests.

[Shabab Hannan]

I recommend reading a recent paper on confined displacement algorithm, by
Ramirez et al., (2010. Journal of Microscopy). this technique allows
analysis of colocalization within a confined area or ROI. It allows to
distinguish between colocalization that is random from which is significant.
The technique has some useful visual tools for demonstration of results and
is easy to apply using the image J plugin named confined displacement
algorithm.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2010.03369.x/abstract

Shabab hannan



On Thu, May 19, 2011 at 2:44 AM, Oleg Broytman <[hidden email]> wrote:

> Greetings.  I need some advice about how to use Image J to carry out my
> colocalization analysis.  I am studying changes in protein localization in
> the Phrenic Motor Nucleus (PMN)- a cluster of neurons in the cervical spinal
> cord.  I have acquired Z-stack images under the confocal microscope of PMN
> neurons.  The PMN neurons are immunostained for Protein 1 on the Green
> channel of fluorescence and Protein 2 on the Red Channel of fluorescence.
> They are also labeled with a tracer dye, the fluorescence of which is
> acquired on the Far Red channel.
>
> What I would like to do is to analyze the extent of colocalization between
> Protein 1 and Protein 2 - not in the entire field, but only in the regions
> positively stained with the tracer dye.  So, I'd like to select regions of
> interest in each slice of the Z-stack in the Far Red channel (which would
> generate a whole list of ROIs for each stack), and then analyze Green/Red
> colocalization within each region of interest.
>
> I could really use some advice on how to do this.  All the colocalization
> plugins I've encountered thus far analyze 2D colocalization in the entire 2D
> image, or 3D colocalization for the entire stack.  Is there a way to do
> colocalization only within a specific region of interest?
>
> Thank you in advance for your help.
>
> Sincerely,
>
> Oleg Broytman
> University of Wisconsin - Madison.
>