RiFRET - an ImageJ plugin for the evaluation of intensity-based FRET images of live cells


  • János Roszik - ,
  • Duarte Lisboa - ,
  • János Szöllõsi - and
  • György Vereb - vereb@med.unideb.hu, Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary
    Please send questions, comments and requests about the program to János Roszik.


RiFRET is an ImageJ plugin which calculates FRET efficiencies pixel-by-pixel based on intensity-based ratiometric FRET (donor, acceptor and sensitized emission) images. The program allows the user to correct for the possible crosstalks and for autofluorescence, furthermore, to calculate FRET for image stacks/series, i.e. for 3D data sets. Semi-automatic processing is also included in this software. The program works with single tiff and tiff/lsm stack images.

If you are using the plugin, please cite the following paper, in which the program and the analysis are described:
Roszik J, Lisboa D, Szollosi J, Vereb G: Evaluation of Intensity-Based Ratiometric FRET in Image Cytometry-Approaches and a Software Solution, Cytometry Part A 2009; 75A:761-767.

Prospective users should read this paper before running the program. It is available at Cytometry Part A.

Terms of Use

  • You can download and use the program for your data analysis freely.
  • You may modify the program for your own personal usage.

Download (version 1.82, 10 Apr 2009)

Download Excel files for alpha factor calculation

iterative approximation method (Method 2 in the paper)
calculation with a set of FRET pair chimeras (Method 3 in the paper)
(Method 1 and the calculation of the ratio of molar absorption coefficients are included in the program.)


  • a computer that runs ImageJ 1.42k (with Java 1.6.0_05) /other versions of 1.42 or higher should also work, but have not been tested with this version/
    (ImageJ can be easily updated with: Plugins->Utilities->Update ImageJ...)
  • screen resolution 1280 x 900 or higher
    (Java window elements can have different size on different platforms/computers. If you cannot see the buttons correctly, please try to increase the window width/height.)


  • Download RiFRET_Plugin.java to IMAGEJ_HOME_DIRECTORY \ plugins
  • select the menu "Plugins" in ImageJ and choose "Compile and Run..."
  • select RiFRET_Plugin.java and click "Open"

Download example images

(cell line: HeLa, donor: CFP (Cerulean), acceptor: YFP (Venus) - we used the C5V and C17V fusion constructs described in Koushik et al., Biophys J 2006;91:L99)

Download stack example
(with the C17V Cerulean-Venus construct)

If you are using the acceptor photobleaching FRET method, we recommend our other FRET plugin AccPbFRET.

Updated: 2018.07.17.

Dear User!


The University of Debrecen considers the protection of personal data and the information provided to be of paramount importance. We hereby inform you that the University of Debrecen has reviewed its processes and incorporated the requirements of GDPR into his own data management and data protection activities, which entered mandatory on may 25,2018. The personal data of the users has been carefully handled by the University of Debrecen, it complies with the applicable data management regulations.Following the requirements of GDPR, we updated our Privacy Statement, which you can access by clicking on the link below: Privacy Policy.

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