Begin forwarded message:
> Date: Wed, 27 May 2009 08:15:29 -0500
> From: Howard Berg <[log in to unmask]>
> 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
Pfotenhauerstrasse 108
01307 DRESDEN
Germany
+49 (0)15114966933 (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 BioImageXD
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