Fluorescence recovery after photobleaching
Fluorescence recovery after photobleaching (FRAP) is a method for determining the kinetics of diffusion through tissue or cells. It is capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells. This technique is very useful in biological studies of cell membrane diffusion and protein binding. In addition, surface deposition of a fluorescing phospholipid bilayer (or monolayer) allows the characterization of hydrophilic (or hydrophobic) surfaces in terms of surface structure and free energy.
Similar, though less well known, techniques have been developed to investigate the 3-dimensional diffusion and binding of molecules inside the cell; they are also referred to as FRAP.
The basic apparatus comprises an optical microscope, a light source and some fluorescent probe. Fluorescent emission is contingent upon absorption of a specific optical wavelength or color which restricts the choice of lamps. Most commonly, a broad spectrum mercury or xenon source is used in conjunction with a color filter. The technique begins by saving a background image of the sample before photobleaching. Next, the light source is focused onto a small patch of the viewable area either by switching to a higher magnification microscope objective or with laser light of the appropriate wavelength. The fluorophores in this region receive high intensity illumination which causes their fluorescence lifetime to quickly elapse (limited to roughly 105 photons before extinction). Now the image in the microscope is that of a uniformly fluorescent field with a noticeable dark spot. As Brownian motion proceeds, the still-fluorescing probes will diffuse throughout the sample and replace the non-fluorescent probes in the bleached region. This diffusion proceeds in an ordered fashion, analytically determinable from the diffusion equation. Assuming a Gaussian profile for the bleaching beam, the diffusion constant D can be simply calculated from:
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