Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

First, when I sent that email on colocalization without software, I  
was actually cleaning out unfinished drafts in my email, with the  
intent of deleting it.  But, this is a topic that always initiates a  
discussion.

As recommended the paper by Boldt and Cordeliere is very good, as is  
Costes et al, 2004 for the most recent papers, both have refs to  
prior work. the former paper nicely summarizes different types of  
describing colocalization.

The confocal will not completely reject out of focus light due to  
residual aberrations in the optical pathway.  The sample is often  
overlooked as an element of the optical pathway and it will often  
contribute to spherical aberration and background.    Deconvolution  
of  confocal stacks will further reduce noise and effects of  
spherical aberration and scattering.  Of course, it is not a  
substitute for proper sample preparation, adequate sampling and a  
well maintained instrument.  Noise in your images will reduce the  
apparent colocalization by inserting random pixels above threshold,  
while factors contributing to background will raise the appearance of  
colocalization since the signals are spreading throughout the image.   
As mentioned in Jeremy's email, a number of other factors need to be  
considered.  Above all, and as well described in Costes etal,  
controls for your labeling are essential to avoid mistaking such  
things as autofluorescence, bleedthrough or non-specific labeling as  
intensities representing components for which you are analysing.  You  
are pushing the limits of optical resolution, so you will need to  
push the limits of good methodology.

Regards,
Glen



Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
[log in to unmask]

************************************************************************ 
******
The box said "Requires WindowsXP or better", so I bought a Macintosh.
************************************************************************ 
******


On Mar 31, 2008, at 8:05 AM, Valeria Berno wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi,
>
> This question just fit in perfectly on what I am trying to find out  
> about
> colocalization.
>
> When and why do I need do deconvolve pictures collected with a  
> confocal in
> order to be sure about my colocalization (or not colocalization)  
> results?
>
> To be specific: I am working on pre and post-synaptic proteins.
>
> Thanks
>
> Valeria
>
>
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Colocalization based upon "yellow" could be accurate, if and only if,
>> the intensities are comparable and pixel (voxel) quantities in the
>> suspected colocalized volumes are in roughly equal.  .  Otherwise,
>> the yellow is masked by the predominate channel.  Something small,
>> like lysosomes, would need to be sampled properly. Colocalization
>> could be masked by blur unless deconvolved, even if images are
>> collected with a confocal.
>> On Feb 7, 2007, at 1:05 PM, Marc Thibault wrote:
>>
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> Hi all,
>>>
>>> It seems that in many papers from biologists or chemists, and i'm
>>> talking
>>> high impact factors journals,  colocalisation of two elements is is
>>> often
>>> assumed  by simple color superposition (ex: red and green fluoresce
>>> yellow
>>> when colocalising), while microscopists (many physisists I suppose)
>>> seem to
>>> need a more complex software-based confirmation.
>>> Is it ok, when using high end equipment and corrected objectives
>>> (apochromat
>>> with high NA for ex.), to assume colocalisation by color
>>> superposition,
>>> especially when fluorophore are confined to small volume entities,
>>> like
>>> lysosomes ?
>>>
>>> Thanks
>>>
>>> Marc
>>