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AccPbFRET: an ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images.

Roszik J, Szöllosi J, Vereb G - BMC Bioinformatics (2008)

Bottom Line: The 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.The program efficiently speeds up the analysis of large image sets even for novice users and is freely available.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary. janosr@dote.hu

ABSTRACT

Background: The 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.

Results: AccPbFRET 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.

Conclusion: Despite 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.

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Related in: MedlinePlus

Screenshot of an example analysis with the AccPbFRET plugin. 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 also presented.
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Related In: Results  -  Collection

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Figure 1: Screenshot of an example analysis with the AccPbFRET plugin. 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 also presented.

Mentions: The steps of creating the FRET image are enumerated in the main program window, so the user only needs to follow the instructions (see image of main window on the right side of Figure 1 for details). Supplementary information appears as a tooltip when the mouse pointer is hovered over an option or button that might need further explanation. Thus even novice users, those unfamiliar with FRET and/or with Java and ImageJ can quickly go through the analysis procedure, without the danger of committing the usual errors. On average, the complete analysis from loading the images to arriving at reliable results takes less than a minute.


AccPbFRET: an ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images.

Roszik J, Szöllosi J, Vereb G - BMC Bioinformatics (2008)

Screenshot of an example analysis with the AccPbFRET plugin. 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 also presented.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571114&req=5

Figure 1: Screenshot of an example analysis with the AccPbFRET plugin. 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 also presented.
Mentions: The steps of creating the FRET image are enumerated in the main program window, so the user only needs to follow the instructions (see image of main window on the right side of Figure 1 for details). Supplementary information appears as a tooltip when the mouse pointer is hovered over an option or button that might need further explanation. Thus even novice users, those unfamiliar with FRET and/or with Java and ImageJ can quickly go through the analysis procedure, without the danger of committing the usual errors. On average, the complete analysis from loading the images to arriving at reliable results takes less than a minute.

Bottom Line: The 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.The program efficiently speeds up the analysis of large image sets even for novice users and is freely available.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary. janosr@dote.hu

ABSTRACT

Background: The 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.

Results: AccPbFRET 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.

Conclusion: Despite 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.

Show MeSH
Related in: MedlinePlus