FRETcalc � FRET by acceptor photobleaching
|
Author: |
David Stepensky (david.stepensky@yale.edu) from |
|
History: |
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 FRETTH and FRETALL are calculated 2007/06/18: update of the manual |
|
Requires: |
ImageJ 1.31i or later |
|
Limitations: |
Was tested for 8-bit tiff images only, analysis is based on ROI of rectangular shape only. |
|
Source: |
|
|
Installation: |
Download FRETcalc_.java 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. |
|
Description: |
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 v3.0 manual.pdf provides general description of the FRETcalc plugin. FRETcalc_sample.tif contains the images in the required format and could be used for testing and learning the FRETcalc plugin. |