Fluorescence Recovery After Photobleaching (FRAP) is described for the first time by Axelrod et al. (1976).
It is used to measure the rate of lateral diffusion of membrane proteins and lipids.
The technique involves use of fluorescent molecule or fluorophore to label the surface molecules to be analysed.
The Region of Interest (RoI) is photobleached by exposing the region to high intensity laser or to intense light for prolonged period. (Photobleaching or simply bleaching is the process in which fluorophore undergoes irreversible loss of its ability to emit light). The photobleaching of fluorochromes require excitation and presence of molecular oxygen.
The fluorophore is bleached by using light wavelengths that are not absorbed by other molecules of living cells such as enzymes, proteins, nucleic acids. As a result these components of cell will not be affected by photobleaching and we can use the FRAP on living cells without compromising its integrity.
The bleached area slowly recover fluorescence as bleached molecules diffuse away from ROI while fluorescent molecules slowly move in. Hence the technique is called FRAP.
The speed of recovery of photobleach is directly proportional to the rate of lateral diffusion of flourescent molecules.
The photobleaching experiments are conducted by using the confocal laser scanning microscopes. The microscope uses high intensity laser for bleaching and low intensity laser for image recording.
Three different image sequences are obtained after photobleaching;
Prebleach sequence using low intensity laser
Bleach sequence using high intensity laser
Postbleach sequence using low intensity laser
Applications of FRAP:
The technique was originally developed to measure the rate of lateral transport of proteins and lipids in membranes of living cells.
It is nowadays is used to study molecular diffusion, protein dynamics, or interactions between the cellular components.
The binding dynamics between varies cellular components can also be studied by using this technique.
The organization and mobility of lipids in biological membranes can be studied by FRAP.
The cellular transduction and ligand binding sites can be studied using FRAP.
The FRAP can be used to differentiate diffusion from systematic transport processes like electrophoresis, convective flow etc.
The FRAP finds its application in determining the ratio of mobile to immobile components of heterogeneous systems like cell surfaces.
Inverse FRAp (iFRAP):
Was developed by Dundr et al. (2002).
This technique is inverted version of FRAP hence the name iFRAP.
It involves bleaching the entire cell except the region of interest while FRAP involves bleaching the region of interest.
Though the technique is time consuming due to need of bleaching entire cell, it is highly useful
To study diffusion kinetics of molecules
Studying the molecules in small organelles and their interaction with either cytoplasm or other organelles.
Drawbacks:
Since living cells are motile, it is difficult to track region of interest for photobleaching.
Since living cells are three dimensional in nature, FRAP produces 3D cones instead of 2D circles. Due to change in shape of this 3D cone, complex bleached zones are produced.
Fluorophores like GFP exhibit a state of darkness independent of bleaching by laser. So some molecules in ROI can become fluorescent.
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