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Wide-Field FRET Microscopy

Introduction | How to do FRET | Example Images

WIDE-FIELD FRET (W-FRET) MICROSCOPY

Any fluorescence microscope (inverted or upright) can be converted to W-FRET microscopy. There are number of papers listed in the literature for various protein studies using W-FRET system (Day, 1998; Day et al, 2003; Gordon et al, 1998; Jovin and Arndt-Jovin, 1989; Kam et al, 1995; Kraynov et al., 2000; Periasamy and Day, 1999; Varma and Mayor, 1998). For W-FRET it is advisable to use a single dichroic to acquire the donor (D) and acceptor (A) images for the donor excitation wavelength in the double-labeled specimen. This can be achieved by using excitation and emission filter wheels in the microscope system. This option helps to reduce any spatial shift of donor and acceptor channel images, since the processed FRET image is obtained through pixel-by-pixel calculation as described in the FRET data analysis section.

Even though W-FRET microscopy is the simplest and most widely used technique, there is a major limitation to W-FRET in that the emission signals originating from above and below the focal plane contribute to out-of-focus signals that reduce the contrast and seriously degrade the image. Digital deconvolution microscopy in the W-FRET system helps to localize the proteins at different optical sections, but this requires intensive computational process to remove the out-of-focus information from the optical sectioned FRET images (Periasamy and Day, 1998 and 1999). For protein interactions taking place homogeneously over a wider area of a cell (e.g. nucleus), W-FRET is an entirely suitable technique.