FRAP and related optical techniques are the method of choice for observing diffusion in biological systems. FRAP measurements are now normally performed with a laser-scanning confocal microscope (Kubitscheck et al., 1994). The component whose diffusion is to be observed must be tagged with a fluorophore so that it can be imaged in the confocal microscope. To observe the diffusion of the fluorophore, the laser power is increased and the confocal spot is scanned briefly over a small area of the sample, so as to bleach the fluorophore. There are several possible mechanisms of photochemical bleaching, of which the commonest is probably photo-oxidation (Xie and Trautmann, 1998). After bleaching, the laser power is decreased again and the whole sample is imaged. The bleached area of the sample will be seen as a dark, non-fluorescent patch on the image. The sample is repeatedly imaged, and if the fluorophore is mobile the bleaching will change in a characteristic way. As the fluorophore diffuses, the fluorescence in the centre of the bleach will recover, and the bleach will become broader and shallower. In favourable cases, it is then possible to analyse the images to obtain an accurate value for the diffusion coefficient for the fluorophore. The requirements for quantitative FRAP are that the membrane geometry is known, and the membrane environment is uniform over an area considerably larger than the area of the bleach. As FRAP is an optical technique, it has limited resolution (the theoretical limit is about half the wavelength of light being used). When performing FRAP measurements in vivo the resolution may be further reduced by sample scattering, and diffusion of the fluorophore in the short time-interval between bleaching and recording the first post-bleach image. In practice, it was found that the minimum bleach width in this study was about 0.5–1 µm.
Ideal membrane systems for FRAP include the extensive, flat, plasma membranes of cultured fibroblasts (Kubitscheck et al., 1994). Chromophores used for FRAP measurements include native photosynthetic pigments (Mullineaux et al., 1997), GFP (Reits and Neefjes, 2001), and a range of dyes which may be conjugated to proteins or to lipid analogues. The use of GFP-fusions to tag specific proteins for FRAP is becoming routine (Reits and Neefjes, 2001). Photo-activatable forms of GFP have now been developed. In these variants of GFP, exposure to specific wavelengths of light induces a transition to a stable conformational with greatly increased fluorescence yield. This offers the promise of being able to carry out FRAP measurements at lower laser intensities, using the laser to photo-activate GFP and increase fluorescence in a limited region of the sample (Patterson and Lippincott-Schwartz, 2002). It is conceivable that the GFP tag could alter the mobility of the membrane protein, although this is perhaps unlikely, given that diffusion in membranes is generally very much slower than in the cytoplasm (compare Zhang et al., 1993, with Elowitz et al., 1999). Thus, an additional cytoplasmic domain is unlikely to be a significant drag on an integral membrane protein unless it specifically binds to other components in the cytoplasm. Where possible, a functional assay should be carried out on the GFP-tagged transformant to see if the function of the tagged protein is perturbed.
Answers to all your Biology Questions
