Their emission range is such that background fluorescence is often reduced. Even donor-acceptor pairs with separated emission spectra i. Large separation between pairs allows the measurement of acceptor emission as a result of FRET without interference from donor emission. In addition these molecules can be linked directly to specific locations in synthetically produced nucleic acids, allowing FRET to be used to assess nucleic acid annealing.
Figure 4. Schematic representation of FRET occurring between Cy3 and Cy5 fluorescent moieties when labeled oligonucleotides are annealed. In the example depicted in Figure 4, two complementary RNA oligonucleotides are labeled with Cy3 and Cy5 respectively. When these labeled molecules are not annealed Figure 4A , excitation of an RNA oligonucleotide labeled with Cy3 with light at nm results only in the emission of light by Cy3 at nm, while the complementary RNA-oligo labeled with Cy5 does not emit any light at nm or its true emission wavelength of nm.
However, when the two oligonucleotides are allowed to anneal, the close proximity of the molecules allows for FRET transfer to occur.
This results in the emission of light at nm when the annealed molecule is excited with nm light. Note that not all of the emission of Cy3 at nm is lost, but a significant portion is. Figure 5. It is the distance dependence that is utilized with VSPs. Resting cells have a relatively negative potential, so the two probes associate with the exterior of the cell membrane, resulting in efficient FRET.
When the membrane potential becomes more positive, as occurs with cell depolarization, the oxonol probe rapidly translocates on a subsecond time scale to the other face of the membrane Figure 5. Thus, each oxonol probe "senses" and responds to voltage changes in the cell. Lanthanide compounds, such as europium and terbium, have been effectively used as donors in FRET reactions. In addition, the lengthy fluorescent half-life allows for measurement to begin after the cessation of excitatory light.
The delay between excitation and measurement msec range allows for background fluorescence from the organic acceptor molecule, with a half-life in the nanosecond range, to dissipate.
Thus only donor-acceptor emission is measured when the appropriate delay is enabled. The donor molecule need not always involve a fluorescent compound. Luminescent molecules emit photons in a fashion very similar to fluorescence.
The primary difference is that the electron excitation is not the result of photon absorption, but rather from the release of chemical energy contained within the molecule.
When the excited electrons return to their ground energy can be released as a photon of light or transferred via RET to an acceptor molecule if the conditions are correct. While more limited in the number of molecules that can be utilized, this technology has the advantage that there is no external excitation of the acceptor molecule.
When designing FRET experiments there are a number of issues that need to be considered. The most obvious issue is the matter of close proximity. Finally, polarization can be degraded in high numerical aperture objectives, so polarized FRET experiments should be limited to imaging with objectives having a numerical aperture of 1. Presented in Figure 8 is a graphic illustration of polarization anisotropy using fluorescent proteins as a model system. When a randomly oriented population of fluorescent proteins Figure 8 a is excited with linearly polarized light cyan wave , only those molecules whose absorption dipole vector is oriented parallel to the polarization azimuth are preferentially excited.
Emission from properly oriented fluorescent proteins can be observed as a signal using an analyzer that is also parallel to the excitation light polarization vector green wave. The resulting anisotropy, which is an indicator of the degree of orientation, can be determined by measuring and comparing the emission intensity through the vertically and horizontally oriented analyzers.
The anisotropy signal level will decrease if the fluorescent protein rotates in the timescale of the experiment Figure 8 b or if it transfers excitation energy due to FRET to a neighboring protein Figure 8 c having a different orientation. As described above, due to the fact that resonance energy transfer can occur far more rapidly than molecular rotation for large fluorescent protein molecules, depolarization due to FRET can be readily distinguished from the loss of anisotropy that occurs during rotation.
The choice of suitable probes for examining FRET in living cells is limited. Synthetic fluorophores, ideal for resonance energy transfer investigations in fixed cells, are difficult to administer and target in live cells.
Likewise, quantum dots can be utilized to label membrane components for examination of phenomena on the exterior of a cell, but they too are unable to penetrate the membrane and, consequently, of little use in intracellular compartments such as the nucleus, mitochondria, or endoplasmic reticulum.
Genetically encoded fluorescent proteins currently represent the best candidates for high-resolution imaging of FRET in live cells, as evidenced by the volume of literature that is published in this arena on a yearly basis. However, many of the typical artifacts that are encountered in measuring FRET with synthetic fluorophores and quantum dots are particularly acute when applied to fluorescent proteins. For example, contrary to the nanometer bandwidth of emission spectral profiles in synthetics, those in fluorescent proteins range from approximately 60 nanometers to nanometers, often leading to significant overlap when attempting to segregate donor and acceptor fluorescence.
The broad spectra of fluorescent proteins also limit the number of probes that can be used together in FRET and other types of imaging experiments. Furthermore, fluorescent proteins exhibit a wide variation in brightness levels. The ends of the barrel are capped with semi-helical peptide regions that serve to block entry of ions and small molecules. The interior of the protein is so tightly packed with amino acid side chains and water molecules that there is little room for diffusion of oxygen, ions, or other intruding small molecules that manage to pass through the ends of the barrel.
These favorable structural parameters, which are partially responsible for the resilient photostability and excellent performance of fluorescent proteins, also contribute to a reduction in FRET efficiency. The large size of the barrel effectively shields adjacent fluorescent protein chromophores with peptide residues to a limiting close approach distance of 2 to 3 nanometers; indicated by the red line in Figure 9 , resulting in a reduction of the maximum FRET efficiency to approximately 40 percent of the theoretical value.
Regardless, the numerous benefits of using fluorescent proteins for live cell FRET imaging far outweigh the costs. Compounding the high degree of spectral bandwidth overlap and size problems that occur with fluorescent proteins is their tendency to oligomerize. Almost all of the fluorescent proteins discovered to date display at least a limited degree of quaternary structure, as exemplified by the weak tendency of native Aequorea victoria green fluorescent protein and its derivatives to dimerize when immobilized at high concentrations.
This tendency is also verified by the strict tetramerization motif of the native yellow, orange, and red fluorescent proteins isolated in reef corals and anemones.
Oligomerization can be a significant problem for many applications in cell biology, particularly in cases where the fluorescent protein is fused to a host protein that is targeted at a specific subcellular location. Once expressed, the formation of dimers and higher order oligomers induced by the fluorescent protein portion of the chimera can produce atypical localization, disrupt normal function, interfere with signaling cascades, or restrict the fusion product to aggregation within a specific organelle or the cytoplasm.
This effect is particularly marked when the fluorescent protein is fused to partners which themselves participate in natural oligomer formation. Fusion products with proteins that form only weak dimers in effect, most Aequorea victoria variants may not exhibit aggregation or improper targeting, provided the localized concentration remains low.
However, when weakly dimeric fluorescent proteins are targeted to specific cellular compartments, such as the plasma membrane, the localized protein concentration can, in some circumstances, become high enough to permit dimerization. This can be a particular concern when conducting intermolecular FRET experiments, which can yield complex data sets that are sometimes compromised by dimerization artifacts. On the other hand, the naturally occurring weak dimerization in Aequorea proteins can be, in some cases, utilized to increase the FRET signal in biosensors that otherwise would exhibit limited dynamic range.
Toxicity is an issue that occurs due to excessive concentrations of synthetic fluorophores and the over-expression or aggregation of poorly localized fluorescent proteins. Furthermore, the health and longevity of optimally labeled mammalian cells in microscope imaging chambers can also suffer from a number of other deleterious factors.
Foremost among these is the light-induced damage phototoxicity that occurs upon repeated exposure of fluorescently labeled cells to illumination from lasers and high-intensity arc-discharge lamps. In their excited state, fluorescent molecules tend to react with molecular oxygen to produce free radicals that can damage subcellular components and compromise the entire cell. Fluorescent proteins, due to the fact that their fluorophores are buried deep within a protective polypeptide envelope, are generally not phototoxic to cells.
In designing FRET experiments, fluorescent protein combinations that exhibit the longest possible excitation wavelengths should be chosen in order to minimize damage to cells by short wavelength illumination, especially in long-term imaging experiments. Thus, rather than creating fusion products and biosensors with blue or cyan fluorescent proteins excited by ultraviolet and blue illumination, respectively , variants that emit in the yellow, orange, and red regions of the spectrum would be far more ideal.
Investigators should take care to perform the necessary control experiments when using new fluorescent protein biosensors and cell lines to ensure that cytotoxicity and phototoxicity artifacts do not obscure FRET results or other important biological phenomena.
In some cases, lipophilic reagents induce deleterious effects that may be confused with fluorescent protein toxicity during imaging in cell lines following transient transfections.
Oligomeric fluorescent proteins discussed above from reef corals have a far greater tendency to form aggregates combined with poor subcellular localization than do the monomeric jellyfish proteins, but improperly folded fusion products can occur with any variant. Recently, a fluorescent protein capable of generating reactive oxygen species ROS upon illumination with green light has been reported as an effective agent for inactivation of specific proteins by chromophore-assisted light inactivation CALI.
Appropriately named KillerRed , this genetically encoded photosensitizer is capable of killing both bacteria and eukaryotic cells upon illumination in the microscope. Previous studies on EGFP phototoxicity indicate that even through the chromophore is capable of generating singlet oxygen, the fluorescent protein is relatively inefficient as a photosensitizer.
However, prolonged illumination of cells expressing EGFP and its variants can result in physiological alterations and eventual cell death, a definite signal of the potential for phototoxicity in long-term imaging experiments.
In live-cell experiments, fluorescent proteins are highly advantageous for extended imaging due to their reduced rate of photobleaching when compared to synthetic fluorophores. Although there is a high degree of uncorrelated variability between fluorescent proteins in terms of photostability, most variants are useful for short-term imaging from 1 to 25 captures , while several of the more photostable proteins can be employed in time-lapse sequences that span periods of 24 hours or longer in which hundreds to thousands of images are gathered.
The long term stability of any particular protein, however, must be investigated for every illumination scenario widefield, confocal, multiphoton, swept-field, etc. Thus, in terms of photostability, the selection of fluorescent proteins is dictated by numerous parameters, including the illumination conditions, the expression system, and the effectiveness of the imaging setup.
Over the past few years, a wide variety of new fluorescent protein variants have been developed and refined to feature emission profiles spanning a nanometer range from approximately nanometers to nanometers , thus filling many gaps to provide potentially useful FRET partners in every color class.
Recent advances in developing proteins in the blue nanometers to nanometers and cyan nanometers to nanometers spectral regions have yielded several new probes that may be of use for imaging and FRET investigations. Three protein engineering groups have reported improved blue Aequorea fluorescent protein variants that feature significantly higher brightness and photostability compared to EBFP. The brightest and most photostable of the new blue reporters, EBFP2, exhibits typical GFP-like behavior in fusions and has been demonstrated to be an excellent FRET donor for proteins in the green spectral class.
All of the blue fluorescent proteins can be readily imaged in a fluorescence microscope using standard DAPI filter sets or proprietary BFP sets available from aftermarket manufacturers.
Fluorescent proteins in the cyan spectral region have been widely applied as FRET donors when paired with yellow-emitting proteins, and were dominated by variants of the original Aequorea ECFP until the introduction of a monomeric teal-colored reporter, known as mTFP1.
Teal fluorescent protein exhibits higher brightness and acid stability compared to Aequorea CFPs, and is far more photostable.
Additional investigations have produced useful proteins in the cyan spectral class. Among the improved cyan fluorescent proteins that have recently been introduced, CyPet and the enhanced cyan variant termed Cerulean show the most promise as candidates for fusion tags, FRET biosensors, and multicolor imaging. Cerulean is at least 2-fold brighter than ECFP and has been demonstrated to significantly increase contrast as well as the signal-to-noise ratio when coupled with yellow-emitting fluorescent proteins, such as Venus see below , in FRET investigations.
However, CyPet has a more blue-shifted and narrower fluorescence emission peak than CFP, which greatly increases its potential for multicolor imaging.
The introduction of beneficial folding mutations into monomeric variants of ECFP has resulted in the production of new variants featuring enhanced brightness, folding efficient, solubility, and FRET performance. Termed super CFPs SCFPs , the new reporters are significantly brighter than the parent protein when expressed in bacteria and almost two-fold brighter in mammalian cells.
Specific details about the protein are unavailable in the literature, but it is commercially available as mammalian cloning vectors and fusions from Evrogen. This protein features the longest absorption and emission wavelength profiles and nanometers, respectively reported for any probe in the cyan spectral region. The high molar extinction coefficient and quantum yield exhibited by MiCy render the protein of equal brightness to Cerulean. Emerald contains the F64L and S65T mutations featured in EGFP, but the variant also has four additional point mutations that improve folding, expression at 37 degrees Celsius, and brightness.
Recently, a new addition to the green spectral region has been coined superfolder GFP , which is brighter and more acid resistant than either EGFP or Emerald and has similar photostability. Therefore, the superfolder variant should be an excellent candidate for fusions with mammalian proteins and the construction of FRET biosensors, especially those that demonstrate folding problems with standard GFP derivatives.
Another brightly fluorescent reporter, which may be a good FRET candidate, is termed Azami Green and has been isolated from the stony coral Galaxeidae and demonstrated to mature rapidly during expression in mammalian cell lines.
In addition, two bright, monomeric GFP reporters obtained through site-directed and random mutagenesis in combination with library screening in cyan proteins have been reported. Derived from the Clavularia coral genus, mWasabi is a potential alternative green-emitting FRET partner for blue fluorescent proteins due to negligible absorbance at nanometers and lower where blue variants are often excited. The new green reporter is commercially available Allele Biotechnology and should be particularly useful for two-color imaging in conjunction with long Stokes shift proteins such as T-Sapphire as well as a localization tag in fusions with targeting proteins.
A derivative of TagCFP, named TagGFP , is a bright and monomeric green variant having an absorption maximum at nanometers and emission at nanometers. TagGFP, which is only slightly brighter than EGFP, is available as cloning vectors and fusion tags from Evrogen, but has not been thoroughly characterized in literature reports. Yellow fluorescent proteins nanometers to nanometers are among the most versatile genetically-encoded probes yet developed and should provide candidates acting as both donors and acceptors in FRET pairings.
The variants known as Citrine and Venus are currently the most useful proteins in this spectral class see Table 1 , but neither is commercially available. Another variant, named after the birthstone Topaz , is available from Invitrogen and has been of service in fusion tag localization, intracellular signaling, and FRET investigations.
Similar to its partners, TagYFP emission peak at nanometers has not been characterized in the literature, but can be purchased as mammalian cloning vectors or fusion tags. During the same fluorescence-activated cell sorting investigation that led to the generation of CyPet discussed above , the evolutionary optimized complementary FRET acceptor, termed YPet , was also obtained. The resistance to acidic environments afforded by YPet is superior to Venus and other YFP derivatives, which will enhance the utility of this probe in biosensor combinations targeted at acidic organelles.
However, although the optimized CyPet-YPet combination should be the preferred starting point in the development of new FRET biosensors, there remains a serious doubt as to the origin of YPet's increased performance, which is likely due simply to enhanced dimerization with its co-evolved partner, CyPet. Likewise, the suitability of CyPet and YPet in fusion tags for localization experiments, bimolecular complementation analysis, and other applications has yet to be established.
Both proteins exist in solution as weak dimers, but presumably can be converted to true monomers using the AK mutation that has worked so well with other Aequorea variants although this apparently destroys their advantages in FRET. Orange fluorescent proteins, all of which have all been isolated from coral reef species, have the potential to be useful in a variety of FRET imaging scenarios.
Thus, the more overlap of spectra, the better a donor can transfer energy to the acceptor. The resonance energy transfer mechanism is also affected by the orientations of the emission transition dipole of the donor and the absorption dipole of the acceptor. FRET provides an efficient way to measure the distance between a donor and an acceptor chromophore. Thus, by measuring the FRET efficiency, one can easily get the precise distance between the donor and the acceptor.
If choosing the donor and acceptor properly, this experiment can also be carried out in vivo. However, the FRET only gives the information about distances. If a dramatic conformational change happens, such as lengthening or kink, it is unable to know the exact movement of donor and the acceptor.
Besides, attaching the chromophores to precise sites of a macromolecule is also important, both in quantity of chromophores and in position of a macromolecule, or the FRET might produce noise signals. Please refer to question 5. The F-actin filament is composed by G-actin monomers. By attaching either a donor D or an acceptor A choromophore to the G-actin monomer and measuring the energy transfer efficiency to gauge the average distance between G-actin monomers in a F-actin filment assuming that the monomers are well arranged in DADADADA The simplest and the most popular one is the sensitised emission method, where the donor is excited by a specific wavelength of light and the signal is collected by using emission filters chosen for the donor fluorescence and the acceptor fluorescence.
Additionally, this method could be the best option if there is no cross-talk between FRET pairs. Unfortunately cross-talk between fluorophores does exist in the real world and corrective approaches and appropriate controls are required to make this method useful for dynamic experiments in which FRET changes are large.
The acceptor photobleaching method is simple but limited to a single measurement. This method is based on the fact that the donor is quenched when FRET occurs. By photobleaching the acceptor, you release the donor's quenching and the fluorescence of the donor is increased. This method is straightforward and quantitative, but it is destructive and cannot be used for dynamic measurements.
Extra care should be taken so as not to destroy the donor molecule. FLIM measures the fluorescence decay time of the donor. In addition, other environmental factors, such as pH or autofluorescence background, can change the fluorescence decay time and have to be taken into account when interpreting data.
FRET is 20 years old but as you can imagine not old-fashioned at all. Add Comment. Addgene is a nonprofit plasmid repository.
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