PixFRET, an ImageJ plug-in for FRET calculation which can accommodate variations in spectral bleed-throughs

Jérôme Feige1, Daniel Sage2, Walter Wahli1, Béatrice Desvergne1 and Laurent Gelman1

1 - Center for Integrative Genomics, NCCR frontiers in Genetics, University of Lausanne, Switzerland.
2 - Biomedical Imaging Group (BIG), Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.


Fluorescence Resonance Energy Transfer (FRET) is a technique used to investigate interactions between fluorescent partners that has gained great interest for cell biologists with the recent introduction of auto-fluorescent proteins which can be coupled to a protein of interest to produce a fluorescent chimera.

A plethora of methods exists to calculate FRET, depending on the protocol used (e.g. sensitized emission versus acceptor photobleaching) and the precision that is pursued (for a review, see "FRET or no FRET: a quantitative comparison", Biophysical J., 2003:84 p.3992-4010).

The ImageJ plug-in PixFRET allows to generate images of FRET, and hence to visualize FRETwithin a cell or a cell population, by computing pixel by pixel the images of a sample acquired in a three channel setting, according to the formula and the methodology described by Gordon et al (Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy, Biophysical J., 1998:74(5), p.2702-13) and Xia and Liu ("Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes", Biophysical J., 2001:81(4), p.2395-402).

FRET Image Website_1.jpg
ExpNFRET reduces inter-cellular variability in pixel-by-pixel analyses.

Cos-7 cells were transfected with expression vectors for PPARalpha-ECFP and RXRalpha-EYFP. (Upper panel) Images of two cells expressing both PPAR-ECFP and RXR-EYFP in the CFP and YFP settings. A Gaussian blur of 1 was applied to the original image. (Lower panel) NFRET and expNFRET images generated by the PixFRET plug-in. The cells are pseudo-colorized with a different look up table to better visualize intensity differences.


Plug-in installation

  1. Download the file
  2. Double click on the icon to extract the plug-in. This will create a folder with two new files. The ".jar" file is the plug-in and the ".pdf" file the user's guide.
  3. Place the ".jar" file in the "Plugins" folder in ImageJ.
  4. Start ImageJ again to display the new plug-in in the "Plugins" menu.

Note that ImageJ is a public-domain software package for image processing. It is free and it doesn't take more than a couple of minutes to install (http.//, it runs on any platforms: Unix, Linux, Windows, Mac OS9, Mac OSX.

Plug-in and user's guide

Last update of the plug-in : 2006/05/17

 inside this site:
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