Abstract
BackgroundThe acceptor photobleaching fluorescence resonance energy transfer (FRET) method is widely used for monitoring molecular interactions in cells. This method of FRET, while among those with the simplest mathematics, is robust, self-controlled and independent of fluorophore amounts and ratios.ResultsAccPbFRET is a user-friendly, efficient ImageJ plugin which allows fully corrected, pixel-wise calculation and detailed, ROI (region of interest)-based analysis of FRET efficiencies in microscopic images. Furthermore, automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing FRET evaluation software.ConclusionDespite of the widespread applicability of the acceptor photobleaching FRET technique, this is the first paper where all possible sources of major errors of the measurement and analysis are considered, and AccPbFRET is the only program which provides the complete suite of corrections – for registering image pairs, for unwanted photobleaching of the donor, for cross-talk of the acceptor and/or its photoproduct to the donor channel and for partial photobleaching of the acceptor. The program efficiently speeds up the analysis of large image sets even for novice users and is freely available.
Highlights
The acceptor photobleaching fluorescence resonance energy transfer (FRET) method is widely used for monitoring molecular interactions in cells
This latter phenomenon is the basis of the popularity of FRET in biology: The distance over which FRET occurs is small enough to characterize the proximity of possibly interacting molecules, under special circumstances it even provides quantitative data on exact distances, and, information on the spatial orientation of molecules or their domains
FRET can be measured both in microscopic imaging and in flow cytometry
Summary
With our program AccPbFRET, which can be found in the additional file [see Additional file 1] or can be downloaded from its homepage [28], FRET efficiencies are calculated pixel-by-pixel, and their distribution is determined for any user defined rectangular, polygonal, or freehand type ROI or subcellular location. The ImageJ (left top) and AccPbFRET (right) dialog windows are displayed, together with donor channel source images taken by a confocal microscope (labeled appropriately as 'Donor before bleaching', 'Donor after bleaching'), as well as the calculated, corrected FRET image ('Transfer image'), on which ROIs can be selected and statistics calculated. These statistics can be seen in a separate 'Results' window, and a histogram of the FRET distribution is presented. Automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing evaluation software
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