FRAP or Fluorescence Recovery After Photobleaching, is a technique that is used to detect and characterize the mobility of cellular molecules. This technique was first used to analyze the mobility of individual lipid molecules within a cell membrane. This technique is also used to study protein dynamics outside the membrane, which is a region of interest within the cytoplasm or cellular structures within the cell that can be monitored using this technique.
Photobleaching experiments can be conducted with confocal laser scanning microscopes where a high intensity laser beam is used for bleaching and a low intensity laser beam is used for image recording.
The FRAP employs irradiation of a fluorophore in a living sample with a short laser to erase fluorescence which is followed by time-resolved image recording of the sample.
In consequence there are three different image sequences:
1) The prebleach sequence with low laser power setting to have a reference value,
2) The bleach sequence with high laser power inside the ROI
3) The postbleach setting with low laser power setting to examine the recovery of fluorescence.
All other bleaching methods are derived from this basic principle.
The protein of interest is first made fluorescent by expression of a GFP (green fluorescent protein cloned from the jellyfish) fusion protein or by tagging the protein of interest with reactive ligands which then binds a fluorescent dye. The most common way to fluorescently tag proteins in living cells is GFP technology,
The phenomenon of photobleaching which is also commonly referred to as fading, occurs when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification. Upon transition from an excited singlet state to the excited triplet state, fluorophores may interact with another molecule to produce irreversible covalent modifications. The triplet state is relatively more stable compared to the singlet state, thus allowing excited molecules a much longer time period to undergo chemical reactions with components in the environment.
The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent upon the molecular structure and the local environment. Some fluorophores bleach quickly after emitting only a few photons, while others that are more robust can undergo thousands or millions of cycles before bleaching. This interactive tutorial explores variations in photobleaching rates in single, dual, and multiply labeled fluorescence specimens.
The Experimental Setup of FRAP:
The experimental setup of FRAP comprises of:
1) a microscope.
2) a light source.
3) a fluorescent probe coupled to the molecule of interest.
Several images using a low light level are acquired to determine the initial fluorescence, and then a high level of light for a short time inside a region of interest is applied to bleach the fluorescence.
Finally, another set of images using a light level sufficiently low to prevent further bleaching is acquired to gain insight into the redistribution of molecules via recovery of fluorescence
The Uses of FRAP:
1) The technique of FRAP is used to visualize the lateral mobility and dynamics of fluorescent proteins in living cells.
2) Non-invasive fluorescent tagging became possible with the green fluorescent protein (GFP) due to this technique.
3) GFP is used to study the localization of fusion proteins in fixed or living cells.
4) These fluorescent proteins can also be used to study protein mobility in living cells.