- Introduction
Many cellular processes are elegantly studied by biochemistry. Usually, this involves experiments where large number of cells are lysed, and protein content is subsequently isolated and studied using antibodies to detect changes in protein levels, post-translational modifications, pairing with partner molecules, etc. Although extremely informative in many cases, these mass population analyses often lack the time resolution to study rapid alterations in protein state, and do not allow the characterisation of highly dynamic processes. Moreover, analysis of millions of cells at once, evidently show the average response in the population of cells, thereby obscuring cell-to-cell variation and the dynamic range of a process. Finally, subcellular compartmentalisation of reactions is difficult to assess in these whole-cell approaches.
With the availability of microscopic techniques in combination with genetically encoded fluorescent probes, many of the described restraints have been overcome. Highly dynamic reactions can now be studied in detail in a relatively easy manner, and in the context of a living cell.
- Fluorescence Resonance Energy Transfer
One of these techniques is Fluorescence Resonance Energy Transfer, or FRET, a physical phenomenon already described many years ago. FRET is the radiationless transfer of energy from an excited donor fluorophore to a suitable acceptor fluorophore, a physical process that depends on spectral overlap and proper dipole alignment of the two fluorophores (see figure). Whereas normally an excited fluorophore returns back to the ground state with the emission of a photon, FRET results in the excitation of the nearby acceptor fluorophore that in turn emits a photon when it returns to the ground state. The occurrence of FRET is characterized by a decrease in observed donor emission, and a simultaneously sensitized (increased) acceptor emission (see figure). Although described many years ago, FRET has gained renewed interest with the availability of genetically encoded GFP variants (see sidebar).- Distance is everything
Importantly, FRET is extremely sensitive to the distance between the fluorophores (see figure). For CFP and YFP, the characteristic half-maximum distance, or Förster radius, is 49-52Å. Because of this sensitivity, and the fact that these distances perfectly match the sizes of most biological molecules, FRET is an ideal way of studying biological processes (see figure). For example, it can be used to study protein-protein interactions if both molecules are tagged with a fluorophore. FRET in that case will only be observed if both proteins are so close to each other that they almost certainly interact. A second application is to follow conformational changes by tagging a single protein with two fluorophores. Changes in conformation of the protein translate in alteration of FRET between the fluorophores. Finally, similar concepts have been used to successfully construct probes for measuring the presence and concentration of small biomolecules like divalent ions (i.e. calcium) and second messengers (i.e. cyclic AMP), or for measuring activity of enzymes like kinases.- Confocal FRET microscopy
FRET can be detected by measuring sensitized emission through confocal microscopy. This way interaction between proteins or conformational changes can be studied in living cells at unprecedented resolutions. Still, these measurements requires carefully calibrated equipment and many controls. One of the major problems comes from the necessary spectral overlap of CFP and YFP. Although strictly required for FRET to occur, it makes separation of both signals very difficult, and leak-through signals are therefore inevitable. To compensate for leak-through, we co-culture together with our cells of interest, cells that express either CFP or YFP tagged histon 2b (H2B), see figure. Using software developed in our institute by the group of Kees Jalink, the CFP and YFP images are corrected for direct excitation of YFP, and CFP emission leak-through in the YFP channel. The software also corrects for a number of other problems specifically encountered using confocal FRET microscopy (see reference below for detailed information). Next, the separately acquired CFP and YFP images are used after correction to calculate the sensitized emission, or 'FRET' image. This signal is still correlated to the concentration of molecules at a specific location, and only tells whether FRET occurs or not. Therefore, the FRET signal is divided by the amount of donor or acceptor molecules to determine the FRET efficiency at each location. An example experiment is shown here.- Reference
- J. van Rheenen, et al. Biophysical Journal 2004 vol. 86, pp.2517-2529 |
PubMed >>
FRET thanks it recent revival to the Northwest Pacific jellyfish Aequorea victoria known for the green light it emits. In the early 1990s, scientists discovered that this light is produced by two proteins. The first, aequorin produces blue light, which is then converted by the second protein in green light that was accordingly named Green Fluorescent Protein, or GFP. Since then, scientist have created a wide range of color variants. Two of these, CFP (cyan) and YFP (yellow) are perfectly suited for FRET measurements, and generated renewed interest in this technique.
In 1948 Th. Förster published his work Zwischen-molekulare energiewanderung und fluoreszenz, in which he describes that the efficiency of FRET between two fluorophores decreases with their distance to the power of six. This has lead to the term Förster radius which is the distance between two fluorophores at which the FRET efficiency is half-maximal. Because FRET is so sensitive to distance, it can function as a molecular ruler, although in many cases the uncertainty of fluorophore orientation and position prevents absolute measurements.
The lifetime of a fluorophore is the average time it stays in the excited state before it falls back to the ground state, normally in the order of nanoseconds. One of the hallmarks of FRET is a decrease in the lifetime of the donor fluorophore because of the induced transfer of energy to the nearby acceptor. Fluorescence Lifetime Imaging Microscopy, or FLIM, takes advantage of this phenomenon to detect the occurrence of FRET by imaging lifetimes rather than the intensity of fluorophores. One of the big advantages of this technique is that it is considered much more quantitative than classical FRET approaches.