Using color threshold and analyze particles to compare colocalization?

[Mike Holloway]

I'm attempting to compare degrees of colocalization in zebrafish motor
axons.  I've been researching ways to measure colocalization for months now
and I'm still uncertain of the best way to procede.  Read the Coloc2
instructions and accompanying guides, but that software seems designed to
accurately detect colocalization rather than allow comparison of degrees of
colocalization between many control and experimental samples.  If I'm wrong
about that please correct me, but I can't see how the coefficients can be
used in this way, and have failed to find any paper in which they are used
that way.  What I've settled on is masking axons, making a max intensity Z
projection image, isolating a narrow band of colocalized color (yellow) with
color threshold set to default, and using analyze particles to find the
percent of the total area that the colocalized puncta take up.  So far I've
found no difference between my experimental and control which seems to be
telling me that either this method is kosher, or I'm messing up big time.  

 

As I said, if there's something in the ImageJ/Fiji docs that address this
I'm not picking up on it.  Been over them several times.  If anyone has
suggestions, or assurances, please let me know.

 

Thanks,

Mike Holloway


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[gankaku]

Hi Mike,

You could try also the JACoP Plugin and read the corresponding paper (Bolte
S, Cordelieres FP. A guided tour into subcellular colocalization  analysis
in light microscopy. J Microsc. 2006;224:213-32. as well as Dunn et al.,
2011 AJP-CP). Those are very good to understand tha different analyses
(which most are also used by the Coloc2 Plugin) and to figure out which
might be suitable to evaluate co-localization in your case. Those
literature also describes a lot of pitfalls and things to take care of.
Since you mentioned the degree of co-localization the Mander's coefficient
might be especially of interest for you (if applicable to your images).
Obligatory in all cases will be a low background and high
signal-to-noise-ratio. Masking of your structures of interest or spezific
background subtraction methods (Median Background Subtraction from the
BioVoxxel Toolbox update site) might also be an option before using those
analysis tools (as also stated in Dunn et al.).

hope this helps a little bit.
regards,
Jan




2014-05-07 19:52 GMT+02:00 Mike Holloway <[hidden email]>:

> I'm attempting to compare degrees of colocalization in zebrafish motor
> axons.  I've been researching ways to measure colocalization for months now
> and I'm still uncertain of the best way to procede.  Read the Coloc2
> instructions and accompanying guides, but that software seems designed to
> accurately detect colocalization rather than allow comparison of degrees of
> colocalization between many control and experimental samples.  If I'm wrong
> about that please correct me, but I can't see how the coefficients can be
> used in this way, and have failed to find any paper in which they are used
> that way.  What I've settled on is masking axons, making a max intensity Z
> projection image, isolating a narrow band of colocalized color (yellow)
> with
> color threshold set to default, and using analyze particles to find the
> percent of the total area that the colocalized puncta take up.  So far I've
> found no difference between my experimental and control which seems to be
> telling me that either this method is kosher, or I'm messing up big time.
>
>
>
> As I said, if there's something in the ImageJ/Fiji docs that address this
> I'm not picking up on it.  Been over them several times.  If anyone has
> suggestions, or assurances, please let me know.
>
>
>
> Thanks,
>
> Mike Holloway
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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