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Problems with establishing colocalisation by overlaying images using different layers of an RGB image

1) A change to the detector gain and fiddling in Photoshop changes the apparent colocalisation.

2) The appearance of pixels, where the intensities are similar but are low, is highly dependent on the setup of the monitor and on limitations of printing.

3) Interpretation is therefore highly subjective and Journals should demand a higher level of evidence. 

4) Software for quantitation is widely available. Most of which generates a very standard set of measurements. 

It should be recognized that the quantitative and non quantitative methods are dependent upon having images of good quality and that are precisely aligned

    

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Glen MacDonald
Sent: Fri 3/28/2008 22:54
To: [log in to unmask]
Subject: Re: colocalisation without software
 
> 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