- Introduction
Fluorescence Recovery after Photobleaching (FRAP) has been available to scientists for almost three decades already. The technique was developed around 1976 in the lab of Watt W. Webb when they were interested in the lateral mobility of the Acetylcholine Receptor (AChR). The introduction of the Green Fluorescent Protein (GFP), and the development of commercially available confocal-microscope-based photobleaching methods have lead to a revival of the fluorescence technique in the late 1990s. Using this technique, a region of interest is selectively photobleached with a high-intensity laser, and the recovery that occurs as fluorescent molecules move into the bleached region is monitored over time with low-intensity laser light. Depending on the protein studied, fluorescence recovery can result from protein diffusion, binding/dissociation or transport processes.
- Technically explained
A typical Jablonski diagram of a fluorophore (see figure) illustrates a singlet ground electronic state (the parallel bars labeled S0), as well as singlet first (S1; upper set of parallel bars) and sometimes a second electronic excited state (S2; not shown). At each energy level, fluorophores can exist in a number of vibrational energy levels, which are represented by the multiple lines in each electronic state. Transitions between states are depicted by a sphere (representing an electron), which is followed by a vertical line that traverses the region between the ground and excited state. The electronic transitions are almost instantaneous in nature, often occurring in timeframes ranging from nano- to picoseconds, which are far too short to observe significant lateral displacement of nuclei during fluorescence and phosphorescent events. Fluorescence lifetimes are typically four orders of magnitude slower than vibrational relaxation, which is shown as the drop from S1 back to S0.
In photobleaching, the electron undergoes a spin conversion into a "forbidden" triplet state (T1) instead of the lowest singlet excited state, a process known as intersystem crossing. Upon transition from this excited singlet state to the excited triplet state, fluorophores may interact with another molecule (i.e. oxygen) to produce irreversible covalent modifications. The triplet state is relatively long-lived with respect to the singlet state, thus allowing excited molecules a much longer timeframe 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.- Recovery room
Analysis of the fluorescence recovery can be used to determine kinetic parameters of a protein, including its diffusion constant, mobile fraction, transport rate or binding/dissociation rate from other proteins. In short, the fluorescence in the region of interest is measured during the complete protocol; just before, ideally also during, and of course after photobleaching. This will lead to a characteristic recovery curve as shown in the figure. Depending on the complexity of the interactions of the protein of interest, the curve may have different shapes (differences in the steepness of the curve, the plateau level and even multiple plateaus and curves). From a simple single exponential curve one can calculate the mobile fraction (R) with the help of the function: R = (F∞ - F0)/(Fi - F0). The t½ allows comparison of diffusion between different conditions. The t½ is the time that is necessary for the fluorescence to recover half between the fluorescence level after bleaching (F0) and the fluorescence at the plateau level (F∞).- FLIP side
Complementary to the photobleaching techniques discussed earlier, the continuity of a cell compartment can be monitored using a technique called Fluorescence Loss In Photobleaching (FLIP). In a FLIP experiment, a fluorescent cell is repeatedly photobleached within a small region while the whole cell is continuously imaged. Any regions of the cell that are connected to the area being bleached will gradually lose fluorescence due to lateral movement of mobile proteins into this area. By contrast, the fluorescence in unconnected regions will not be affected. In addition to assessing continuity between areas of the cell, FLIP can be used to assess whether a protein moves uniformly across a particular cell compartment or undergoes interactions that impede its motion. See figure for an example with two cells expressing a proteasome subunit tagged with GFP. Even after up to 180 minutes the fluorescence between nucleus and cytoplasm is not equalized, indicating that the proteasome is not diffusing between the two compartments. Finally, FLIP can be used to reveal faint fluorescence in unconnected compartments that normally cannot be seen against the bright fluorescence from other parts of the cell.- Reference
- E. Reits and J. Neefjes, Nature Cell Biology 2001 vol. 3, pp.E145-147 |
From fixed to FRAP >>
- J. Lippincott-Schwarz, et al. Nature Cell Biology 2003, pp.S7-14 | Photobleaching and photoactivation >>
Born in the Ukraine in 1898, Alexander Jablonski (1898-1980) is best known as the father of fluorescence spectroscopy. Most of his life he worked as a scientist in Warsaw, Poland in the Department of Experimental Physics. There he introduced the Jablonski diagrams to depict energy transitions occuring in fluorophores in 1935.
Confocal is defined as "having the same focus". What this means in the microscope is that focus of the image corresponds to the point of focus in the object. The object and its image are "confocal." Normally when an object is imaged in the fluorescence microscope, the signal produced is from the full thickness of the specimen which does not allow most of it to be in focus to the observer. The confocal microscope eliminates this out-of-focus information by means of a confocal "pinhole" situated in front of the image plane which acts as a filter and allows only the in-focus portion of the light to be imaged. Light from above and below the plane of focus of the object is eliminated from the final image. To make this all possible, a strong excitation source is required. Modern confocal microscopes use scanning lasers to excite the sample.
If one lab has made an extraordinary and unique contribution to the development of a series of methods and techniques for measuring and visualising processes within and between cells, then it is the lab of Roger Tsien in San Diego, California, USA. Since the early 1980s this lab has made great efforts in the field of non-invasive tools for measuring calcium fluxes, protein interactions and protein visualization. For his work in this field he has been awarded multiple prices, including the Dutch Dr H.P. Heineken Prize for Biochemistry and Biophysics 2002.
Photo-activatable GFP, or PA-GFP, was developed with the aim of optimizing the photoconversion properties of Aequorea victoria wtGFP, which produces only a threefold increase in fluorescence under 488 nm excitation. Mutation of threonine 203 to histidine decreases the initial absorbance in the minor peak region (475 nm) and leads to 100-fold increase after photoactivation. The ability to 'switch on' the fluorescence of the photoactivatable proteins makes them excellent tools for exploring protein behavior in living cells. As the fluorescence of these proteins is present only after photoactivation, newly synthesized non-photoactivated pools are unobserved and do not complicate experimental results. The method can also be used as a reciprocal of FRAP.
Some processes in the cell are fast, really fast. To measure these you need a really fast system. The conventional, commercially available, confocal microscopes are often too slow to accurately record these processes, with fluorescence already fully recovered before the laser has switched from bleaching to imaging mode. One solution is the linescan FRAP with external bleaching laser. A single line is scanned continuously with high frequency, while the external laser bleaches the region of interest with milliseconds pulses. The result is an xt-plot from which recovery curves in the milliseconds timeframe can be derived, fast enough to follow soluble GFP for example.