FRETcalc – FRET by acceptor photobleaching


David Stepensky ( from Ben-Gurion University of the Negev, Israel


2006/09/05: Version 1.0
2006/11/01: Version 2.0 - more output options added
2007/05/08: Version 3.0 - change in the calculation algorithm, both FRETTHand FRETALL are calculated
2007/06/18: update of the manual
2010/09/19: Version 4.0 - All types of regions of interest (polygonal, rectangular, elliptical, and freehand ROIs) are accepted
2012/03/06: Version 5.0, the bug with arguments calling was fixed (thank to Robert W. Baer and Johannes Schindelin).


ImageJ 1.31i or later


Was tested for 8-bit tiff images only





Download to the plugins folder, or subfolder, and compile and run using Plugins/Compile and Run. Restarting ImageJ will add a "FRETcalc" command to the Plugins menu or a submenu of the Plugins menu.


FRETcalc plugin for ImageJ program allows pixel-by-pixel analysis of FRET by acceptor photobleaching. FRETcalc uses thresholding FRETTH algorithm to exclude from data analysis pixels that do not match user-defined thresholds of donor and/or acceptor fluorescence intensity, bleaching efficiency, and %FRET. Subsequently, FRET signal-containing pixels on the analyzed images could be separated from the fluorophore-free background pixels, making possible analysis of FRET signal arising from non-continuous intracellular organelles/compartments (e.g., endoplasmic reticulum, endosomal system, etc.). Effect of the threshold-based pixel selection on the estimated FRET values can be determined by comparing FRETTH and FRETALL (that is calculated using all the pixels in the selected region of interest (ROI)). The plugin allows easy visualization of the analysis results (as plots and/or histograms), thus helping to estimate the statistical robustness of FRET analysis. FRETcalc plugin, its validation, and application to study protein-protein interactions in the MHC class I loading complex in the endoplasmic reticulum has been described in: Stepensky D. FRETcalc plugin for calculation of FRET in non-continuous intracellular compartments. Biochem Biophys Res Commun 2007; 359(3):752-8.
FRETcalc v5.0 manual.pdf provides general description of the plugin.
FRETcalc_sample.tif file contains the images in the required format and could be used for testing and learning the FRETcalc plugin.

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