Re: colocalization question

Posted by Cameron Nowell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/colocalization-question-tp2272746p2277469.html

Howdy,

          Sorry to chip in a bit late to this discussion but one thing you need to always remember with colocalisation of fluorecent images, that is resolution. Sorry to those that already know all this but is is an important point to emphasise when talking about colocalisation.

 

Resolution is governed by NA and can be calculated using 0.61 x emission wavlength/NA (for widfield) and 0.4 x emision wavelength/NA (for confocal). There are a few variations of this formula, and while they give theoretical best resolutions, the resolution acheived in practice is usually always worse. Also this is only for lateral resolution, axial resolution is worse by a facotr of about 3.

 

So for a 60x 1.4NA objective on a confocal your best lateral resolution for say GFP is about 150nm (220nm for widefield). That means that your proteins could be 200nm apart and still "colocalise". 200nm (2000 angstroms) is a rather large distance when you are talking about protein interactions. For example a human herpes virus is aorund 200nm across, there are a lot of proteins in that small particle, but not all of them are directly interating with each other.

 

Normally when people are doing colocalisation they have immunoprecipitation or other such data to show the proteins they are interested in interact, couple this with the fluorecent colocalisation and it is much more convincing. The other choice is FRET imaging, as FRET requires proteins to be 1-10nm appart for it to occur. So even though you can't resolve the protein with a confocal you can indirectly measure its interaction.

 

If you want an accurate image to show proteins interacting you have to use immuno EM as EM can give you the resolution you need.

 

Sorry for the rant :)

 

 

Cam

 

 

Cameron J. Nowell
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jean-Pierre CLAMME
Sent: Friday, 6 February 2009 3:57 AM
To: [hidden email]
Subject: Re: colocalization question

Thank you all very much. Your inputs  helped a lot !

JP

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Jean-Pierre CLAMME, PhD
Senior Scientist
Nitto Denko Technical
501 Via Del Monte
Oceanside, CA 92058
E-mail: [hidden email]
Phone: [phone]


Confocal Microscopy List <[hidden email]> wrote on 02/05/2009 03:00:41 AM:

> Dear JP,

> You need to measure thresholded Manders coefficients (1 for red , 1
> for green!)

> search this list archives for my previous long posts on this topic.

>
> in short, use ImageJ or easier BioImageXD (both free)
> to calculate threshold using the methods of costes,
> then calculate the Manders coefficents 1 and 2
> then do the costes statistical significance test.

> Publish:
> thresholds, manders coefficents, statistical significance,
> 2D histogram . scatter plots with straight line fit and thresholds.

> You need to read 2 papers at least
> Costes et al
> and Manders et al.

> I will send them to you in a moment

> cheers

> Dan

>
> Begin forwarded message:

> >

> > Date:    Wed, 4 Feb 2009 17:05:30 -0800
> > From:    Jean-Pierre CLAMME <[hidden email]>
> > Subject: colocalization question
> >
> > This is a multipart message in MIME format.
> > --=_alternative 0005FF9888257554_=
> > Content-Type: text/plain; charset="US-ASCII"
> >
> > Hi,
> >
> > When you do colocalization measurement (in image pro for example)
> > between
> > let say the red and a green channel the co-localization coefficient
> > tells
> > you how much red and green are co-localized.  But as much as I
> > understood,
> > it doesn't give you information on the fact that for example red is
> > always
> > co-localized with green but green is not always colocalized with red ?
> > What kind of analysis  would I need to compare this between samples ?
> >
> > Thank you in advance,
> >
> > JP
> >
> >
> > - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
> > Jean-Pierre CLAMME, PhD
> > Senior Scientist
> > Nitto Denko Technical
> > 501 Via Del Monte
> > Oceanside, CA 92058
> > E-mail: [hidden email]
> > Phone: [phone]

> Dr. Daniel James White BSc. (Hons.) PhD
> Senior Microscopist / Image Processing and Analysis
> Light Microscopy Facility
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany

>
> New Mobile Number!!!

> [phone] (German Mobile)
> +49  (0)351 210 2627 (Work phone at MPI-CBG)
> +49  (0)351 210 1078 (Fax MPI-CBG LMF)

> http://www.bioimagexd.net
> http://www.chalkie.org.uk
> [hidden email]
> ( [hidden email] )

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