Limits...
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.

Show MeSH

Related in: MedlinePlus

The analysis process. In this figure, the changes of donor and acceptor images during the steps of the analysis process are shown. DB, DA: donor images before and after bleaching the acceptor; AB AA: acceptor images before and after bleaching (same images as in Figure 1, cropped to fit the page). STEP1: original images; STEP2: images after registration (note the disappearance of the top lines from DA and AA); STEP3: after background subtraction; STEP4: after Gaussian filtering; STEP 5: thresholded images. The corrected FRET/transfer image and histogram derived from it are also displayed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2571114&req=5

Figure 2: The analysis process. In this figure, the changes of donor and acceptor images during the steps of the analysis process are shown. DB, DA: donor images before and after bleaching the acceptor; AB AA: acceptor images before and after bleaching (same images as in Figure 1, cropped to fit the page). STEP1: original images; STEP2: images after registration (note the disappearance of the top lines from DA and AA); STEP3: after background subtraction; STEP4: after Gaussian filtering; STEP 5: thresholded images. The corrected FRET/transfer image and histogram derived from it are also displayed.

Mentions: Figure 2 shows the images as they evolve through the analysis process to finally yield the FRET ("transfer") image. As a biological example, intramolecular FRET characteristic of receptor conformation was measured among two cell surface ErbB2 tyrosine kinase epitopes on SK-BR-3 cells using a 4-channel CLSM. The two epitopes were labeled specifically by the fluorescently tagged antibodies rHu4D5 (trastuzumab) and 2C4. Confocal imaging was carried out with a Zeiss (Göttingen, Germany) LSM 510 confocal laser scanning microscope (CLSM) using a Plan-Apochromat 63×/NA 1.4, oil DIC objective. The donor, AlexaFluor 555 was excited with a 543-nm HeNe laser and detected through a 560–615 nm emission filter. As acceptor, Cy5 was excited with a 633-nm HeNe laser and detected through a 650 nm longpass filter. The panels in the four consecutive rows depict donor (D) and acceptor (A) channel images before (DB, AB) and after (DA, AA) photobleaching the acceptor. The columns show the original images (step 1); images after registration (step 2); after background subtraction (step 3); after Gaussian filtering (step 4); and finally the thresholded images (step 5). Correction factors are obtained using a similar algorithm. The corrected FRET/transfer image is then calculated, and the histogram derived from it is also displayed. The measurement in this case reveals a mean FRET efficiency in the cell membrane of 14.5%, indicating that the extended dimerization loop of ErbB2 is in proximity of the juxtamembrane domain. Such measurements can be specific enough to support molecular modeling as was demonstrated in the case of the nearly full length ErbB2 earlier [29].


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

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

The analysis process. In this figure, the changes of donor and acceptor images during the steps of the analysis process are shown. DB, DA: donor images before and after bleaching the acceptor; AB AA: acceptor images before and after bleaching (same images as in Figure 1, cropped to fit the page). STEP1: original images; STEP2: images after registration (note the disappearance of the top lines from DA and AA); STEP3: after background subtraction; STEP4: after Gaussian filtering; STEP 5: thresholded images. The corrected FRET/transfer image and histogram derived from it are also displayed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The analysis process. In this figure, the changes of donor and acceptor images during the steps of the analysis process are shown. DB, DA: donor images before and after bleaching the acceptor; AB AA: acceptor images before and after bleaching (same images as in Figure 1, cropped to fit the page). STEP1: original images; STEP2: images after registration (note the disappearance of the top lines from DA and AA); STEP3: after background subtraction; STEP4: after Gaussian filtering; STEP 5: thresholded images. The corrected FRET/transfer image and histogram derived from it are also displayed.
Mentions: Figure 2 shows the images as they evolve through the analysis process to finally yield the FRET ("transfer") image. As a biological example, intramolecular FRET characteristic of receptor conformation was measured among two cell surface ErbB2 tyrosine kinase epitopes on SK-BR-3 cells using a 4-channel CLSM. The two epitopes were labeled specifically by the fluorescently tagged antibodies rHu4D5 (trastuzumab) and 2C4. Confocal imaging was carried out with a Zeiss (Göttingen, Germany) LSM 510 confocal laser scanning microscope (CLSM) using a Plan-Apochromat 63×/NA 1.4, oil DIC objective. The donor, AlexaFluor 555 was excited with a 543-nm HeNe laser and detected through a 560–615 nm emission filter. As acceptor, Cy5 was excited with a 633-nm HeNe laser and detected through a 650 nm longpass filter. The panels in the four consecutive rows depict donor (D) and acceptor (A) channel images before (DB, AB) and after (DA, AA) photobleaching the acceptor. The columns show the original images (step 1); images after registration (step 2); after background subtraction (step 3); after Gaussian filtering (step 4); and finally the thresholded images (step 5). Correction factors are obtained using a similar algorithm. The corrected FRET/transfer image is then calculated, and the histogram derived from it is also displayed. The measurement in this case reveals a mean FRET efficiency in the cell membrane of 14.5%, indicating that the extended dimerization loop of ErbB2 is in proximity of the juxtamembrane domain. Such measurements can be specific enough to support molecular modeling as was demonstrated in the case of the nearly full length ErbB2 earlier [29].

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