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.
- You can download and use the program for your data analysis freely.
- You may modify the program for your own personal usage.
Download (version 1.83, 24 May 2010):
- RiFRET_Plugin.java (Java source code)
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/
(Version 1.42k ij.jar can be downloaded from here.)
- 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 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):
- 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"
If you are using the acceptor photobleaching FRET method, we recommend our other FRET plugin AccPbFRET.