Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

Abstract

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...

Efeitos dos corticoesteroides nas lesões por radiofrequência na coxa de ratos em diferentes faixas etárias

“…In the present paper, on the other hand, for the first time in the literature, the lesions were measured through specific software [6][7][8][9][10][11][12] , confirming that the corticosteroids reduce the lesion area in the infants and pubescents. These results can not be attributed to individual variations or to discrepancies in the ablation procedure.…”

“…For the planimetric evaluation of the lesions, ImageJ™ 6,7 software was used -broadly disseminated in cytometry 8 and other planimetric studies [9][10][11][12] -which automatically calculates the lesion area ( Figure 1) with good precision and measurement converter to any desired unit by the operator. The measurement is manual, using the computer mouse that, contouring the edges of the lesion, generates automatically the measurement result.…”

rFRET: A comprehensive, Matlab‐based program for analyzing intensity‐based ratiometric microscopic FRET experiments

“…The measured intensities (I 1-3 ) are described by the following set of equations and references (2,5,6):…”

“…Solving the above equation set for the FRET efficiency (E) yields different results depending on which overspill constants can be neglected and on the form of the equation describing I 3 . The solutions have been published elsewhere (2,(5)(6)(7)(8).…”

“…While some of them are aimed at Technical Note high-throughput evaluation without allowing the user much control over the calculations (7), others require more userinput to fine-tune the evaluation (6,9). The software tool presented in the current manuscript belongs to the second group performing scientifically rigorous and fully overspill-corrected calculations with many user-adjustable options.…”