Not Available in Your Country
Fluorescence Resonance Energy Transfer (FRET) Microscopy

The precise location and nature of the interactions between specific molecular species in living cells is of major interest in many areas of biological research, but investigations are often hampered by the limited resolution of the instruments employed to examine these phenomena. Conventional widefield fluorescence microscopy enables localization of fluorescently labeled molecules within the optical spatial resolution limits defined by the Rayleigh criterion, approximately 200 nanometers (0.2 micrometer). However, in order to understand the physical interactions between protein partners involved in a typical biomolecular process, the relative proximity of the molecules must be determined more precisely than diffraction-limited traditional optical imaging methods permit. The technique of fluorescence resonance energy transfer (more commonly referred to by the acronym FRET), when applied to optical microscopy, permits determination of the approach between two molecules within several nanometers (see Figure 1), a distance sufficiently close for molecular interactions to occur.

Typical fluorescence microscopy techniques rely upon the absorption by a fluorophore of light at one wavelength (excitation), followed by the subsequent emission of secondary fluorescence at a longer wavelength. The excitation and emission wavelengths are often separated from each other by tens to hundreds of nanometers. Labeling of cellular components, such as the nuclei, mitochondria, cytoskeleton, Golgi apparatus, and membranes, with specific fluorophores enables their localization within fixed and living preparations. By simultaneously labeling several sub-cellular structures with individual fluorophores having separated excitation and emission spectra, specialized fluorescence filter combinations can be employed to examine the proximity of labeled molecules within a single cell or tissue section. With this technique, molecules that are closer together than the optical resolution limit appear to be coincident, and this apparent spatial proximity implies that a molecular association is possible. In most cases, however, the normal diffraction-limited fluorescence microscope resolution is insufficient to determine whether an interaction between biomolecules actually takes place. Fluorescence resonance energy transfer is a process by which radiationless transfer of energy occurs from an excited state fluorophore to a second chromophore in close proximity. Because the range over which the energy transfer can take place is limited to approximately 10 nanometers (100 angstroms), and the efficiency of transfer is extremely sensitive to the separation distance between fluorophores, resonance energy transfer measurements can be a valuable tool for probing molecular interactions.

The mechanism of fluorescence resonance energy transfer involves a donor fluorophore in an excited electronic state, which may transfer its excitation energy to a nearby acceptor chromophore in a non-radiative fashion through long-range dipole-dipole interactions. The theory supporting energy transfer is based on the concept of treating an excited fluorophore as an oscillating dipole that can undergo an energy exchange with a second dipole having a similar resonance frequency. In this regard, resonance energy transfer is analogous to the behavior of coupled oscillators, such as a pair of tuning forks vibrating at the same frequency. In contrast, radiative energy transfer requires emission and reabsorption of a photon and depends on the physical dimensions and optical properties of the specimen, as well as the geometry of the container and the wavefront pathways. Unlike radiative mechanisms, resonance energy transfer can yield a significant amount of structural information concerning the donor-acceptor pair.

Resonance energy transfer is not sensitive to the surrounding solvent shell of a fluorophore, and thus, produces molecular information unique to that revealed by solvent-dependent events, such as fluorescence quenching, excited-state reactions, solvent relaxation, or anisotropic measurements. The major solvent impact on fluorophores involved in resonance energy transfer is the effect on spectral properties of the donor and acceptor. Non-radiative energy transfer occurs over much longer distances than short-range solvent effects, and the dielectric nature of constituents (solvent and host macromolecule) positioned between the involved fluorophores has very little influence on the efficacy of resonance energy transfer, which depends primarily on the distance between the donor and acceptor fluorophore.

The phenomenon of fluorescence resonance energy transfer is not mediated by photon emission, and furthermore, does not even require the acceptor chromophore to be fluorescent. In most applications, however, both donor and acceptor are fluorescent, and the occurrence of energy transfer manifests itself through quenching of donor fluorescence and a reduction of the fluorescence lifetime, accompanied also by an increase in acceptor fluorescence emission. The efficiency of the energy transfer process varies in proportion to the inverse sixth power of the distance separating the donor and acceptor molecules. Consequently, FRET measurements can be utilized as an effective molecular ruler for determining distances between biomolecules labeled with an appropriate donor and acceptor fluorochrome when they are within 10 nanometers of each other.

A hypothetical example of fluorescence resonance energy transfer between two fluorochromes attached to opposite ends of the same macromolecular protein is presented in Figure 1. In the native conformation (Figure 1(a)), the two fluorophores are separated by a distance of approximately 12 nanometers, too far for intramolecular resonance energy transfer between the fluorochromes to occur. However, when the protein is induced to undergo a conformational change (Figure 1(b)), the two fluorochromes are brought much closer together and can now participate in FRET molecular interactions. In the figure, excitation of the donor fluorochrome is indicated by a blue glow around the yellow tri-nuclear aromatic molecule, while the corresponding acceptor emission (Figure 1(b)) is represented by a green glow surrounding the second heterocyclic fluorochrome on the right-hand side of the protein. Energy transfer measurements are often employed to estimate the distances between sites on a macromolecule and the effects of conformational changes on these distances. In this type of experiment, the degree of energy transfer is used to calculate the distance between the donor and acceptor and obtain structural information about the macromolecule.

Although fluorescence resonance energy transfer has often been employed to investigate intermolecular and intramolecular structural and functional modifications in proteins and lipids, a major obstacle to implementation of FRET microscopy techniques in living cells has been the lack of suitable methods for labeling specific intracellular proteins with appropriate fluorophores. Cloning of the jellyfish green fluorescent protein (GFP) and its expression in a wide variety of cell types has become a critical key to developing markers for both gene expression and structural protein localization in living cells. Several spectrally distinct mutation variants of the protein have been developed, including a fluorescent protein that emits blue light (blue fluorescent protein, BFP). Both the excitation and emission spectra for the native GFP and BFP mutants are sufficiently separated in wavelength to be compatible with the FRET approach. Figure 2 illustrates the strategy for detection of protein-protein interactions using fluorescence resonance energy transfer and mutant fluorescent proteins. If two proteins, one labeled with BFP (the donor) and the other with GFP (the acceptor), physically interact, then increased intensity at the acceptor emission maximum (510 nanometers) will be observed when the complex is excited at the maximum absorbance wavelength (380 nanometers) of the donor. Failure of the proteins to form a complex results in no acceptor (GFP) fluorescence emission.

Coupled with advances in pulsed lasers, microscope optics, and computer-based imaging technology, the development of labeling techniques in which the donor and acceptor fluorophores are actually part of the biomolecules themselves has enabled the visualization of dynamic protein interactions within living cells. In addition to the investigation of protein partner interactions, recent applications of fluorescence resonance energy transfer include studies of protease activity, alterations in membrane voltage potentials, calcium metabolism, and the conduction of high-throughput screening assays, such as for quantification of gene expression in single living cells.

Principles of Fluorescence Resonance Energy Transfer

The process of resonance energy transfer (RET) can take place when a donor fluorophore in an electronically excited state transfers its excitation energy to a nearby chromophore, the acceptor. In principle, if the fluorescence emission spectrum of the donor molecule overlaps the absorption spectrum of the acceptor molecule, and the two are within a minimal spatial radius, the donor can directly transfer its excitation energy to the acceptor through long-range dipole-dipole intermolecular coupling. A theory proposed by Theodor Förster in the late 1940s initially described the molecular interactions involved in resonance energy transfer, and Förster also developed a formal equation defining the relationship between the transfer rate, interchromophore distance, and spectral properties of the involved chromophores.

Resonance energy transfer is a non-radiative quantum mechanical process that does not require a collision and does not involve production of heat. When energy transfer occurs, the acceptor molecule quenches the donor molecule fluorescence, and if the acceptor is itself a fluorochrome, increased or sensitized fluorescence emission is observed (see Figure 3). The phenomenon can be observed by exciting a specimen containing both donor and acceptor molecules with light of wavelengths corresponding to the absorption maximum of the donor fluorophore, and detecting light emitted at wavelengths centered near the emission maximum of the acceptor. An alternative detection method, growing rapidly in popularity, is to measure the fluorescence lifetime of the donor fluorophore in the presence and absence of the acceptor.

Presented in Figure 3 is a Jablonski diagram illustrating the coupled transitions involved between the donor emission and acceptor absorbance in fluorescence resonance energy transfer. Absorption and emission transitions are represented by straight vertical arrows (green and red, respectively), while vibrational relaxation is indicated by wavy yellow arrows. The coupled transitions are drawn with dashed lines that suggest their correct placement in the Jablonski diagram should they have arisen from photon-mediated electronic transitions. In the presence of a suitable acceptor, the donor fluorophore can transfer excited state energy directly to the acceptor without emitting a photon (illustrated by a blue arrow in Figure 3). The resulting sensitized fluorescence emission has characteristics similar to the emission spectrum of the acceptor.

Several criteria must be satisfied in order for resonance energy transfer to occur. In addition to the overlapping emission and absorption spectra of the donor and acceptor molecules, the two involved fluorophores must be positioned within a range of 1 to 10 nanometers of each other. As described in equations derived by Förster (and discussed below), the energy transfer efficiency between donor and acceptor molecules decreases as the sixth power of the distance separating the two. Consequently, the ability of the donor fluorophore to transfer its excitation energy to the acceptor by non-radiative interaction decreases sharply with increasing distance between the molecules, limiting the FRET phenomenon to a maximum donor-acceptor separation radius of approximately 10 nanometers. At distances less than 1 nanometer, several other modes of energy and/or electron transfer are possible. The distance dependence of the resonance energy transfer process is the primary basis for its utility in investigation of molecular interactions. In living cell studies involving molecules labeled with donor and acceptor fluorophores, resonance energy transfer will occur only between molecules that are close enough to interact biologically with one another.

An additional requirement for resonance energy transfer is that the fluorescence lifetime of the donor molecule must be of sufficient duration to permit the event to occur. Both the rate (K(T)) and the efficiency (E(T)) of energy transfer are directly related to the lifetime of the donor fluorophore in the presence and absence of the acceptor. According to Förster's theory, and verified experimentally, the rate of energy transfer is given by the equation:

KT = (1/τD) • [R0/r]6

where R(0) is the Förster critical distance, τ(D) is the donor lifetime in the absence of the acceptor, and r is the distance separating the donor and acceptor chromophores. The Förster critical distance (R(0)) is defined as the acceptor-donor separation radius for which the transfer rate equals the rate of donor decay (de-excitation) in the absence of acceptor. In other words, when the donor and acceptor radius (r) equals the Förster distance, then the transfer efficiency is 50 percent. At this separation radius, half of the donor excitation energy is transferred to the acceptor via resonance energy transfer, while the other half is dissipated through a combination of all the other available processes, including fluorescence emission.

Conceptually, the Förster critical distance is the maximal separation length between donor and acceptor molecules under which resonance energy transfer will still occur. The critical distance value typically falls within a range of 2 to 6 nanometers, which is fortuitously on the order of many protein molecular dimensions. In addition, the critical distance range also corresponds to several other biologically significant dimensions, such as cell membrane thickness and the distance separating sites on proteins having multiple subunits. The value of R(0) (in nanometers) may be calculated from the following expression:

R0 = 2.11 × 10-2 • [κ2 • J(λ) • η-4 • QD]1/6

in which κ-squared is a factor describing the relative orientation in space between the transition dipoles of the donor and acceptor, J(λ) is the overlap integral in the region of the donor emission and acceptor absorbance spectra (with the wavelength expressed in nanometers), η represents the refractive index of the medium, and Q(D) is the quantum yield of the donor.

The efficiency of energy transfer, E(T), is a measure of the fraction of photons absorbed by the donor that are transferred to the acceptor, and is related to the donor-acceptor separation distance, r, by the equation:

r = R0 • [(1/ET) - 1]1/6

and E(T) is evaluated as:

ET = 1 - (τDAD)

where τ(DA) is the donor lifetime in the presence of the acceptor and τ(D) is the donor lifetime in the absence of the acceptor. Therefore, by measuring the donor fluorescence lifetime in the presence and absence of an acceptor (which is indicative of the extent of donor quenching due to the acceptor), it is possible to determine the distance separating donor and acceptor molecules. In many commonly applied techniques, the energy transfer efficiency is determined by steady state measurements of the relative average donor fluorescence intensities in the presence and absence of the acceptor (not by measuring the lifetimes).

In summary, the rate of energy transfer depends upon the extent of spectral overlap between the donor emission and acceptor absorption spectra (see Figure 4), the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipole moments, and the distance separating the donor and acceptor molecules. Any event or process that affects the distance between the donor and acceptor will affect the resonance energy transfer rate, consequently allowing the phenomenon to be quantified, provided that artifacts can be controlled or eliminated.

Presented in Figure 4 are the absorption and emission spectra of cyan fluorescent protein (CFP, the donor) and red fluorescent protein (RFP or DsRed, the acceptor) when compared for their potential application as a fluorescence resonance energy transfer pair. Absorption spectra for both biological peptides are illustrated as red curves, while the emission spectra are presented as blue curves. The region of overlap between the donor emission and acceptor absorption spectra is represented by a gray area near the base of the curves. Whenever the spectral overlap of the molecules is increased too far, a phenomenon known as spectral bleed-through or crossover occurs in which signal from the excited acceptor (arising from excitation illumination of the donor) and the donor emission are detected in the acceptor emission channel. The result is a high background signal that must be extracted from the weak acceptor fluorescence emission.

The basic theory of non-radiative energy transfer is directly applicable to a donor-acceptor pair separated by a fixed distance, in which case the rate of energy transfer is a function of the Förster distance, R(0), which in turn depends upon κ-squared, J(λ), η, and Q(D). If these factors are known, the distance between the donor and acceptor can be calculated. More complex formulations are required to describe situations such as multiple acceptor chromophores and distance distributions. Presented in Table 1 are a series of experimentally measured Förster critical distances, which were ascertained from the spectral overlap of several popular donor-acceptor fluorophore pairs. Because the variable includes the donor quantium yield and the degree of spectral overlap, both of which depend on the localized environmental conditions, Förster distance values should be determined under identical experimental conditions as those employed to investigate resonance energy transfer.

The refractive index of the energy transfer medium is generally known from the solvent composition, or can be estimated for a particular macromolecule, and is commonly taken to be 1.4 in aqueous solution. The quantum yield of the donor is determined by comparison to standard fluorophores with known quantum yield. Because Q(D) appears as the sixth-root in the calculation of R(0), small errors or uncertainties in the value of Q(D) do not have a large effect on the Förster distance calculation. Also due to the sixth-root dependence, R(0) is not largely affected by the variations in J(λ), but the overlap integral must still be evaluated for each donor-acceptor pair. In general, higher degrees of overlap between the donor emission spectrum and the acceptor absorption spectrum yield larger Förster critical distance values.

Förster Critical Distance
for Common RET Donor-Acceptor Pairs
Donor
Acceptor
Förster Distance
(Nanometers)
Tryptophan
Dansyl
2.1
IAEDANS (1)
DDPM (2)
2.5 - 2.9
EBFP
DsRFP
3.1 - 3.3
Dansyl
FITC
3.3 - 4.1
Dansyl
Octadecylrhodamine
4.3
ECFP
EGFP
4.7 - 4.9
CF (3)
Texas Red
5.1
Fluorescein
Tetramethylrhodamine
4.9 - 5.5
Cy3
Cy5
>5.0
EGFP
EYFP
5.5 - 5.7
EGFP
Alexa Fluor 546
5.7
EGFP
Alexa Fluor 555
6.3
EGFP
Alexa Fluor 568
5.4
EGFP
Alexa Fluor 594
5.3
BODIPY FL (4)
BODIPY FL (4)
5.7
Rhodamine 6G
Malachite Green
6.1
FITC
Eosin Thiosemicarbazide
6.1 - 6.4
B-Phycoerythrin
Cy5
7.2
Cy5
Cy5.5
>8.0
(1) 5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid
(2) N-(4-dimethylamino-3,5-dinitrophenyl)maleimide
(3) carboxyfluorescein succinimidyl ester
(4) 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

Sorry, this page is not
available in your country.

This site uses cookies to enhance performance, analyze traffic, and for ads measurement purposes. If you do not change your web settings, cookies will continue to be used on this website. To learn more about how we use cookies on this website, and how you can restrict our use of cookies, please review our Cookie Policy.

OK