Photobleaching acceptor

Valentin, G., Verheggen, C., Piolot, T., Neel, H., Coppey-Moisan, M. and Bertrand, E. (2005). Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments. Nat. Methods 2, 801. 

Several other approaches to solve the quantitation problem have been proposed. Hoppe et al. [2] determined y/ by calibrating it against constructs with known FRET efficiency. We and others [3, 6] have used data from a cell before and after acceptor photobleaching to relate the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel by factors termed or G, respectively. For the CFP/YFP pair this works very well on confocal microscopes with a 514-nm Argon ion laser line, but on wide-held systems, selective acceptor photobleaching reportedly causes problems [ 14]. F inally, G can also be determined by comparison of several constructs that differ in FRET efficiency, a bit analogous to the Yellow Cameleon calibration described above [10,14],... 

Gu, Y., Di, W. L., Kelsell, D. P. and Zicha, D. (2004). Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing. J. Microsc. 215, 162-73. 

Donor fluorescence recovery after acceptor photobleaching (also called acceptor photobleaching or acceptor depletion FRET) [22, 23, 26, 28, 30, 48, 53-56] (see Chapters 1 and 7) and... 

A critical prerequisite for acceptor photobleaching is that the acceptor should be readily photobleachable to achieve nearly 95-100% acceptor bleaching in a relatively short period of time. While... 

Fig. 10.3. Acceptor photobleaching analysis of interaction between barley MLO and calmodulin. Barley MLO is a plant-specific integral membrane protein that associates with the cytosolic calcium sensor protein Calmodulin...

Karpova, T. S., Baumann, C. T., He, L., Wu, X., Grammer, A., Lipsky, P., Hager, G. L. and McNally, J. G. (2003). Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J. Microsc. 209, 56-70. 

Van Munster, E. B., Kremers, G. J., Adjobo-Hermans, M. J. and Gadella, T. W., Jr. (2005). Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching. J. Microsc. 218, 253-62. 

Rodighiero, S., Bazzini, C., Ritter, M., Furst, J., Botta, G., Meyer, G. and Paulmichl, M. (2008). Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching. Cell Physiol. Biochem. 21, 489-98. 

In this chapter, we demonstrate the implementation of the acceptor photobleaching FRET method, which is the simplest, robust image cytometric FRET technique that can be performed using any confocal microscopes (15). It involves measuring the donor fluorescence in the presence of the acceptor, and then repeating the measurement after having photobleached the acceptor fluorophore. The result is independent of donor and acceptor concentration and stoichiometry and requires relatively simple image mathematics. 

Acceptor photobleaching FRET measurements can be efficiently analyzed using the AccPbFRET Image plugin (24). The newest version of the program can be freely downloaded from its homepage (25). 

The necessity for using correction factors (apart from that for unwanted donor photobleaching and incomplete acceptor photobleaching) can be avoided by selecting suitable fluorophores. In the case of the AlexaFluor 546-AlexaFluor 647 donor-acceptor pair, it is usually sufficient to correct for unwanted donor photobleaching, and therefore to analyze only the donor images. 

Fig. 2. Anaiysis of images using the AcePbFRET program. This figure shows the main window of the imaged plugin AcePbFRET which is used to analyze acceptor photobleach-ing FRET images. The analysis consists of calculation of correction factors which is performed once at the beginning, followed by seven steps. Those have to be done with all FRET images of the donor and acceptor-labeled samples.

Although low expression levels limit the application of FRET methods, acceptor photobleaching FRET can be successfully used with proteins expressed at as low as 100-150 thousand per cell. 

Roszik J, Szollosi J, Vereb G (2008) AccPbFRET an ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images. BMC Bioinformatics 9 346... 

It is commonly accepted that the most reliable way to measure FRET in cells is the acceptor-photobleaching technique. A donor image is recorded, then the acceptor is destroyed by photobleaching, and another donor image is recorded. The FRET efficiency is obtained from the relative increase of the donor fluorescence intensity [205]. The drawback of the technique is that it is destructive. It is therefore impossible to run successive FRET measurements in the same cell. It is also difficult to use in living cells because the acceptor recovers after photobleaching by diffusion effects. 

Acceptor photobleaching, a method based on quenching donor fluorescence. Some donor photons are used to excite... 

FLIM is not the only technique for observing FRET. If both donor and acceptor chromophores are fluorophores, FRET can also be estimated with steady state techniques, including acceptor photobleaching [102,103], spectral imaging [61,104] and ratio-imaging (or filter-FRET) [105,106] techniques. Recently all possible modes of FRET-microscopy (also including yet unexplored FRET-imaging modes) have been reviewed by Erijman and Jovin [107]. 

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