GFP/YFP colocalization?

[Daniel James White]

Begin forwarded message:

> Date:    Wed, 27 May 2009 08:15:29 -0500
> From:    Howard Berg <[hidden email]>
> Subject: GFP/YFP colocalization?
>
> Hi all:
>
> A paper for ''The Plant Cell'' (Raffaele et al.) just came online and
> several figures are presented showing colocalization of GFP and YFP-
> tagged proteins.  I am not surprised that they colocalized because I
> expect there would be a severe bleedthrough for these two.  Am I
> missing something here or have they presented an artifact?  Their
> methods state that they used sequential acquisition (on a Leica TCS
> SP2).
>
> Howard
>
>

If one looks at the 2D histogram  / scatter plot / fluorogram of the
2 channels compared to each other

(see colocalisation threshold or manders coefficient plugins for image  
J,
or do it in a few clicks in BioImageXD)

then you can see bleed through / cross talk immediately by the
data not following the x and or y axes, but having a gradient offset,
(means where ever you see so much of one dye, you see a proportional  
amount of the other dye... which hints at
the probelm of bleed  through etc.

See slide 33 and surropunds of
https://info.med.tu-dresden.de/MTZimaging/images/2/25/White_QuantitativeColocAnalysis0408a.pdf

cheers
Dan
Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
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