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Figure 2.12.2 A typical recording of a FRAP measurement. The fluorescence intensity (F0) measured in a small region of the sample (typically 3 to 4 |im in diameter) is proportional to the average concentration of fluorochrome in that region. At t = 0, the small region is illuminated with a strong light pulse (bleaching). The fluorochrome in the region undergoes an irreversible, light-induced photo-decomposition and the post-bleaching fluorescence intensity drops to a lower level (F(0)). The fluorescence intensity shows a recovery because intact fluorochrome molecules diffuse into the bleached area due to the Brownian motion. The F(~) intensity of full recovery at long time is lower than F° if the fluorochrome-labeled diffusing compound is partly immobile.

Time

Figure 2.12.2 A typical recording of a FRAP measurement. The fluorescence intensity (F0) measured in a small region of the sample (typically 3 to 4 |im in diameter) is proportional to the average concentration of fluorochrome in that region. At t = 0, the small region is illuminated with a strong light pulse (bleaching). The fluorochrome in the region undergoes an irreversible, light-induced photo-decomposition and the post-bleaching fluorescence intensity drops to a lower level (F(0)). The fluorescence intensity shows a recovery because intact fluorochrome molecules diffuse into the bleached area due to the Brownian motion. The F(~) intensity of full recovery at long time is lower than F° if the fluorochrome-labeled diffusing compound is partly immobile.

such as single-molecule detection sensitivity and high spatial resolution beyond the diffraction limit of the light combined with simultaneous temporal resolution in the millisecond range (Garcia-Parajo et al., 1999).

Revealing the importance of mobility characteristics of molecules and particles. Since its development almost 25 years ago, the FRAP technique has contributed significantly to our understanding of the dynamics of membrane lipids and proteins. The fluid-mosaic model of the cell membrane (Singer and Nicolson, 1971) inspired numerous experimental and theoretical studies to determine the diffusion coefficient and the mobile fraction in natural membranes and model systems. The measurements yielded estimates for the diffusion constant of proteins ranging from D ~ 1-2 x 10-9 cm2/sec to D ~ 10-12 cm2/sec. Perhaps the two most important observations were that (1) the diffusion constant (D) of membrane proteins was considerably lower than expected theoretically from the hydro-dynamic model of Saffman and Delbrück (1975); and (2) the diffusible fraction of proteins (O) was generally <100%. Lipid mobility in the plasma membrane was found to be virtually complete over the distance of FRAP resolution (a few micrometers) with a diffusion constant on the order of D ~ 10-8 cm2/sec. Although early FRAP experiments were not able to detect submicroscopic lipid inhomogeneities, the measurements suggested that the lipids in membranes were not mixed homogeneously but rather segregated in domains (Wolf et al., 1981; Edidin, 1993, 1997). It was concluded that protein diffusion, which is too slow to be determined by lipid viscosity, is somewhat restricted by interactions between membrane proteins and coupling or anchoring to the cytoskeletal or other immobile structural element (Tank et al., 1982; Wu et al., 1982).

Image Cytometry Instrumentation

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