questions about FRET - colcoalization - FLIM

[Daniel James White]

Hi Ramana,

regarding colocalization,
you might like to use the relatively robust methods of Manders and  
Costes for pixel intensity correlation over space.

you can do it with
Fiji (is just imageJ - batteries included) which has working  
colocalisation threshold and Test plugins
http://pacific.mpi-cbg.de

or
BioImageXD
http://www.bioimagexd.net

both for free!

mopst commercial packages dont impement these methods fully.
Auto  threshold is very important,m as is the statistical test to see  
iof the correlation you see is better then random.

See my previous posts on this subject... there is a lot to get right.
Have a look here for a practical guide
http://pacific.mpi-cbg.de/wiki/index.php/ColocalizationAnalysis

and here for theory
https://info.med.tu-dresden.de/MTZimaging/images/2/25/White_QuantitativeColocAnalysis0408a.pdf

and here for quantitative  data collection ideas
https://info.med.tu-dresden.de/MTZimaging/images/6/63/White_QuantitativeColocAnalysis0408b.pdf



By the way..... the most reliable way to do FRET is to actually to do  
FLIM, fluorescence life time imaging.
It gives the information you want, but gets around many of the  
technical problems of traditional FRET that make results unreliable.


cheers

Dan








Hi-

I am interested in looking at colocalization and/or FRET between two
proteins. Unfortunately, the commercially available antibodies for both
proteins are not very good, and the antibodies that have been  
generated by
our group and others that do work well are both in rabbit. I was  
wondering
if there was a good protocol or generally accepted mechanism for dealing
with this.



Thanks,

Ramana Sidhaye
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

[phone] (German Mobile)
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http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de Fiji Fiji is just ImageJ - Batteries Included
http://www.chalkie.org.uk
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