The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results. ' 2009 International Society for Advancement of Cytometry Key terms fluorescence resonance energy transfer; ratiometric FRET; intensity-based FRET; FRET calibration; confocal laser scanning microscopy; ImageJ; 3D image processing IN biological systems, fluorescence resonance energy transfer (FRET) is a widely used method for studying molecular interactions and processes (1). FRET is a nonradiative transfer of energy from an excited fluorophore, the donor, to another fluorophore, the acceptor, that are at a distance of 1-10 nm (2). The efficiency of FRET (E) depends on the inverse sixth power of the distance between the donor and the acceptor, so monitoring E can serve as a spectroscopic ruler (3). Several spectroscopic parameters (e.g., intensity, lifetime, anisotropy) can be used to determine E (1,4-8). In flow cytometry, the intensity-based ratiometric FCET (flow cytometric FRET) method provides statistics for large cell populations. On the other hand, when subcellular details are needed, various microscopic FRET methods can be used (9-16). The ratiometric method (10), which was adapted from flow cytometry to microscopy (17), has the advantage of preserving the donor and acceptor dyes, which is necessary for following the time course of molecular interactions in living cells. Although the ratiometric approach is similar to the 3-cube FRET method (14), it uses less complicated calculations.The achievable signal to noise ratio is usually limited by the autofluorescence of the cells. Since autofluorescence is less pronounced toward the red spectral region, the preferred donor acceptor dye pair should be red-shifted as well, such as the AlexaFluor546 -AlexaFluor647 pair with 543 nm and 633 nm laser excitations, respectively. In a...

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In March 2005, U.S. authorities informed the European Commission of the inadvertent release of unauthorized maize GM event Bt10 in their market and subsequently the grain channel. In the United States measures were taken to eliminate Bt10 from seed and grain supplies; in the European Union an embargo for maize gluten and brewer's grain import was implemented unless certified of Bt10 absence with a Bt10-specific PCR detection method. With the aim of assessing the validity of the Bt10 detection method, an in-depth analysis of the molecular organization of the genetic modification of this event was carried out by both the company Syngenta, who produced the event, and the European Commission Joint Research Centre, who validated the detection method. Using a variety of molecular analytical tools, both organizations found the genetic modification of event Bt10 to be very complex in structure, with rearrangements, inversions, and multiple copies of the structural elements (cry1Ab, pat, and the amp gene), interspersed with small genomic maize fragments. Southern blot analyses demonstrated that all Bt10 elements were found tightly linked on one large fragment, including the region that would generate the event-specific PCR amplicon of the Bt10 detection method. This study proposes a hypothetical map of the insert of event Bt10 and concludes that the validated detection method for event Bt10 is fit for its purpose.