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9/30/2014 Olympus Microscopy Resource Center | Fluorescence Resonance Energy Transfer (FRET) Microscopy - Introductory Concepts 1/12
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Fluorescence Resonance Energy Transfer (FRET) Microscopy
Introductory Concepts
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
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resonance energy transfer, which depends primarily on the distance between the donor and acceptor
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
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 Frster in the late 1940s initially described the molecular interactions involved in
resonance energy transfer, and Frster also developed a formal equation defining the relationship
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between the transfer rate, interchromophore distance, and spectral properties of the involved
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
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 Frster (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 Frster's theory, and verified experimentally, the rate of energy
transfer is given by the equation:
= (1/
) [R
where R(0) is the Frster critical distance, (D) is the donor lifetime in the absence of the acceptor, and
r is the distance separating the donor and acceptor chromophores. The Frster 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 Frster 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 Frster 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
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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:
= 2.11 10
in which -squared is a factor describing the relative orientation in space between the transition dipoles of
the donor and acceptor, J() is the overlap integral in the region of the donor emission and acceptor
absorbance spectra (with the wavelength expressed in nanometers), 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
r = R
) - 1]
and E(T) is evaluated as:
= 1 - (
where (DA) is the donor lifetime in the presence of the acceptor and (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 Frster distance, R(0),
which in turn depends upon -squared, J(), , 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 Frster 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, Frster distance values should be determined under identical experimental conditions as those
employed to investigate resonance energy transfer.
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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 Frster distance calculation. Also due
to the sixth-root dependence, R(0) is not largely affected by the variations in J(), 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 Frster critical distance
Frster Critical Distance for
Common RET Donor-Acceptor Pairs
Donor Acceptor
Frster Distance
Tryptophan Dansyl 2.1
IAEDANS (1) DDPM (2) 2.5 - 2.9
BFP DsRFP 3.1 - 3.3
Dansyl FITC 3.3 - 4.1
Dansyl Octadecylrhodamine 4.3
CFP GFP 4.7 - 4.9
CF (3) Texas Red 5.1
Fluorescein Tetramethylrhodamine 4.9 - 5.5
Cy3 Cy5 >5.0
GFP YFP 5.5 - 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
Table 1
The uncertainty in evaluating the orientation factor (-squared) has been discussed extensively in the
literature, and in spite of experimental evidence that the Frster theory is valid and applicable to distance
measurement, this variable has continued to be somewhat controversial. It is important to recognize that
Frster distances are usually given for an assumed value of -squared, typically the dynamically averaged
value of 2/3 (0.67). This assumed value results from randomization of donor and acceptor orientation by
rotational diffusion prior to energy transfer. The orientation factor depends upon the relative orientations
in space of the donor emission dipole and the acceptor absorption dipole, and can range from zero to 4. A
value of 1 corresponds to parallel transition dipoles, while a value of 4 results from dipoles that are both
parallel and collinear.
Because of the sixth-root relationship to the Frster distance, a variation in the orientation factor from 1 to
4 produces only a 26 percent change in the calculated distance, and a maximum error of 35 percent is
possible when the customarily assumed value of 0.67 is applied. The most serious potential error results if
the dipoles are oriented exactly perpendicular to each other and the corresponding -squared value
becomes equal to zero. Several techniques for dealing with the uncertainty have been employed, including
the assumption that a range of static orientations exist and do not change during the excited-state lifetime
of the fluorophore. Measurements of fluorescence anisotropy for the donor and acceptor can allow limits
to be determined for -squared variation. In addition, utilization of fluorophores with low fluorescence
polarization (due to emission from several overlapping transitions) reduces the uncertainty in orientation
factor. Limitation of the possible values of -squared in this fashion reduces the potential distance
calculation error to perhaps 10 percent.
In many cases, the orientation factor is difficult, if not impossible, to determine and the exact value of the
variable is often regarded as an insurmountable problem. However, some evidence indicates a limitation
for the importance of the factor in resonance energy transfer calculations. Comparison of donor and
acceptor distances using resonance energy transfer spectroscopy and x-ray diffraction techniques largely
supports the validity of assuming a value of 0.67 for the factor (as proposed by Frster theory), at least for
small peptides and proteins. More uncertainty exists for larger proteins. The use of this value for the
orientation factor is valid under the assumption that both donor and acceptor probes are free to undergo
unrestricted isotropic motion. Further justification is gained from experimental evidence that for
fluorophores attached by a single or double bond to macromolecules, segmental motions of the donor and
acceptor tend to result in dynamically randomized orientations.
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For loosely bound fluorochromes, free rotational motion around single bonds should enable use of an
average orientation value, but unrestricted motion of molecules bound through multiple linkage sites
probably does not occur. On the other hand, the extreme values of zero and 4 for -squared require
complete fluorescence polarization of the donor and acceptor, a condition that is unlikely to be achieved.
Statistical calculations have been presented by some investigators that argue the donor-acceptor
distribution distances and their orientations determine the observed average distance. Provided that there
is some distribution in observed distance (and this is not limited by the donor and acceptor being too close
relative to R(0)), the average distance between fluorophores can be reliably obtained and the uncertainty
due to orientation factor assessed.
The dependence of the orientation factor (-squared) on the relative orientations of the donor emission
dipole and the acceptor absorption dipole (illustrated in Figure 5) is given by the equation:

= (cos
- 3cos
= (sin
cos - 2cos
where (T) is the angle between the emission transition dipole of the donor and the absorption transition
dipole of the acceptor, (D) and (A) are the angles between these dipoles and the vector joining the
donor and acceptor, and is the angle between the planes containing the two transition dipoles.
Energy transfer efficiency is most sensitive to distance changes when the donor-acceptor separation
length approaches the Frster distance (R(0)) for the two molecules. Figure 6 illustrates the exponential
relationship between transfer efficiency and the distance separating the donor and acceptor. The
efficiency rapidly increases to 100 percent as the separation distance decreases below R(0), and
conversely, decreases to zero when r is greater than R(0). Because of the strong (sixth-power)
dependence of transfer efficiency on distance, measurements of the donor-acceptor separation distance
are only reliable when the donor and acceptor radius lies within the Frster distance by a factor of two.
When r is approximately 50 percent of R(0), the resonance energy transfer efficiency is near the maximum
and shorter distances cannot be reliably determined. When the donor-acceptor distance exceeds the R(0)
value by 50 percent, the slope of the curve is so shallow that longer separation distances are not
The practical significance of knowing the critical Frster distance is that the value provides a guide to the
range of separation distances that can be determined by FRET for a given pair of probes (see Table 1).
Because energy transfer measurement is very sensitive to distance variation when donor-acceptor
distances are close to the Frster distance, the approximate dimensions of the target molecular interaction
is the most important factor in selecting a fluorescent dye pair. Other factors that should be considered,
depending upon whether steady state or time-resolved measurements are being conducted, include
chemical stability, quantum yield, and fluorophore decay lifetimes. Since no internal distance reference
exists for common fluorescence resonance energy transfer techniques, distances calculated by measuring
transfer efficiencies are relative to a Frster distance, which is derived from spectroscopic data measured
on the donor-acceptor pairs.
The phenomenon of resonance energy transfer by the Frster mechanism is complex in some aspects,
but simple and dependable in its resulting effect. Frster distances are accurately predictable from
spectral properties of the donor and acceptor, and since no exceptions to the theory have yet been
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identified, resonance energy transfer can be assumed to occur under any conditions that place the donor-
acceptor molecule pair in close proximity. The complexity in the theory describing dipole transfer arises,
not because of the transfer mechanism itself, but because of the occurrence of distance distributions
(including nonrandom distributions), and diffusion of the donor and acceptor molecules. When steps are
taken to average the distance dependence of the energy transfer over a range of geometries and
timeframes, FRET is a reliable technique for study of the spatial distributions between interacting
Application of FRET Techniques in Optical Microscopy
Microscope configurational parameters for fluorescence resonance energy transfer investigations vary
with the requirements of the fluorophores, specimen, and imaging mode(s), but virtually any upright or
inverted microscope can be retrofitted for FRET microscopy (see Figure 7). In general, the microscope
should be equipped with a high-resolution (12-bit) cooled and intensified CCD camera system coupled to
quality interference filters having low levels of crosstalk (minimum blocking level) and bandpass regions
corresponding closely to the fluorophore spectra. The detector sensitivity determines how narrow the filter
bandpass can be and still enable data acquisition to proceed at acceptable speeds with a minimum of
spectral bleed-through noise. In most cases, a single dichromatic mirror coupled to excitation and emission
filter wheels or sliders should be used to acquire images in order to minimize or eliminate image shifts.
Widefield fluorescence microscopy suffers from fluorophore emission originating above and below the
focal plane to yield images with significant out-of-focus signal that reduces contrast and leads to image
degradation. This problem is compounded in FRET microscopy because of the inherently low signal levels
produced as a result of resonance energy transfer. Digital deconvolution techniques can be coupled to
optical sectioning in order to reduce or eliminate signals away from the focal plane, but the process is
computationally intensive and may not be fast enough for many dynamic FRET imaging experiments.
Laser scanning confocal techniques can be applied to FRET microscopy to produce a significant
improvement in lateral resolution, while enabling the collection of serial optical sections at intervals
approaching real time. The major drawback of confocal microscopy is the limitation of excitation
wavelengths to the standard laser lines available for a particular system, which restricts the choice of
fluorophore donor and acceptor pairs in resonance energy transfer experiments. Multiphoton excitation
can also be employed in combination with FRET techniques and is less damaging to cells due to the
longer excitation wavelengths involved. In addition, autofluorescence artifacts and photobleaching of the
specimen are less likely to occur within the restricted excitation volume characteristic of multiphoton
A typical microscope configuration capable of observing living cells in culture with several fluorescence
resonance energy transfer imaging motifs is presented in Figure 7. The inverted tissue culture microscope
is equipped with a standard tungsten-halogen lamphouse on the pillar in order to examine and record the
cells using standard brightfield, phase contrast, or differential interference contrast (DIC) illumination. Note
that the latter two contrast enhancing techniques can be employed in combination with fluorescence to
reveal the spatial location of fluorophores within the cellular architecture. A standard Peltier-cooled CCD
camera system is attached to the microscope trinocular head for widefield fluorescence and brightfield
image capture.
Resonance energy transfer experiments are conducted with the multispectral microscope illustrated in
Figure 7 using either widefield illumination (arc discharge lamp) or a real-time scanning confocal
attachment equipped with a high-speed Nipkow disk system. The argon-krypton laser beam is first filtered
through an acousto-optic tunable wavelength device to select specific excitation wavelengths before
passing to the confocal scan head. Images are collected using two high-resolution Gen III-intensified
cooled CCD cameras reading separate channels and spooled to a host computer. Scanning the specimen
in the lateral (x and y) and axial (z) planes enables collection of optical sections for three-dimensional
image reconstruction. A variety of image processing software programs are compatible with the illustrated
microscope configuration.
Based upon the fundamental principles of the phenomenon, a number of important practical points should
be considered when fluorescence resonance energy transfer measurements are conducted with an optical
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The concentrations of donor and acceptor fluorophores must be closely controlled. The
statistically highest probability of achieving fluorescence resonance energy transfer occurs when
a number of acceptor molecules surround a single donor molecule.
Photobleaching must be eliminated because the artifact can alter the donor-to-acceptor
molecular ratio, and therefore, the measured value of the resonance energy transfer process.
The donor fluorescence emission spectrum and the acceptor absorption spectrum should have a
substantial overlap region.
There should be minimal direct excitation of the acceptor in the wavelength region utilized to
excite the donor. A common source of error in steady state FRET microscopy measurements is
the detection of donor emission with acceptor filter sets.
The emission wavelengths of both the donor and acceptor must coincide with the maximum
sensitivity range of the detector.
The donor absorption and emission spectra should have a minimal overlap in order to reduce
the possibility of donor-to-donor self-transfer.
The donor molecule must be fluorescent and exhibit sufficiently long lifetime in order for
resonance energy transfer to occur.
The donor should exhibit low polarization anisotropy to minimize uncertainties in the value of the
orientation (-squared) factor. This requirement is satisfied by donors whose emission results
from several overlapping excitation transitions.
When using antibody labeling techniques, reagents conjugated with donor and acceptor
fluorochromes should not be altered in their biological activity. Any reduction in activity will
seriously affect the validity of resulting resonance energy transfer measurements.
Because fluorescence resonance energy transfer requires the donor and acceptor molecules to
have the appropriate dipole alignment and be positioned within 10 nanometers of each other, the
tertiary structure of the reagents to which the molecules are attached must be considered. For
example, when donor-acceptor molecules can be attached to different structural locations (such
as the carboxy or amino terminus) on a protein, it is possible that FRET will not be observed
even though the proteins do interact, because the donor and acceptor molecules are located on
opposite ends of the interacting molecules.
Living cells labeled with green fluorescent protein mutants for FRET investigations should be
analyzed using traditional immunohistochemical techniques to verify that the tagged protein
adopts the same intracellular habitat and properties as the native counterpart.
In order for the fluorescence resonance energy transfer phenomenon to provide meaningful data as a tool
in optical microscopy, both specimen preparation and imaging parameters must be optimized. The
selection of appropriate donor and acceptor probes and the manner in which they are employed as
molecular labels is a major challenge. In addition, once a labeling strategy that permits energy transfer has
been elucidated, a wide spectrum of techniques may be used to perform the measurement itself. A
majority of quantitative fluorescence microscopy investigations are conducted by measuring the intensity
of fluorescence emission. Fluorescence intensity-based detection of FRET is typically achieved by
monitoring changes in the relative amounts of emission intensity at the two wavelengths corresponding to
the donor and acceptor chromophores. When conditions are appropriate for fluorescence resonance
energy transfer to occur, an increase in acceptor emission (I(A)) is accompanied by a concomitant
decrease in donor emission (I(D)) intensity.
Although a change in the relative emission intensity of either donor or acceptor can be taken as indicative
of resonance energy transfer, the customary approach is to utilize the ratio of the two values, I(A)/I(D), as
a measure of FRET. The value of the ratio depends upon the average distance between donor-acceptor
pairs and is insensitive to differences in the path length and volume accessed by the exciting light beam.
Any specimen condition that induces a change in the relative distance between the molecular pairs
produces a change in the ratio of donor and acceptor emission. Consequently, FRET can be observed in
the microscope by preferential excitation of a donor fluorophore and detection of the increased emission
of an interacting acceptor fluorophore, accompanied by a reduction in donor fluorescence produced by
quenching due to energy transfer. Measurement of FRET employing the intensity-monitoring approach is
termed steady state fluorescence resonance energy transfer imaging.
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Appropriate donor and acceptor probes are selected on the basis of their absorption and emission
spectral characteristics. For maximum resonance energy transfer, the donor emission spectrum should
substantially overlap the absorption spectrum of the acceptor. In addition, there should be minimal direct
excitation of the acceptor fluorophore at the excitation maximum of the donor, and there should not be
significant emission overlap between the donor and acceptor in the wavelength region at which acceptor
emission occurs. In practice, it can be difficult to identify donor-acceptor pairs that satisfy these
requirements. The situation is often complicated by the fact that the commercially available fluorescence
filter sets are not completely effective in passing only the desired wavelengths, and a small percentage of
light outside the design passband may be transmitted. Unless very well characterized and controlled
expression systems are used, the precise concentration of the donor and acceptor fluorophores may be
difficult to determine. Additional corrections may be also be required for autofluorescence,
photobleaching, and background fluorescence.
A typical investigation of intracellular protein-protein association in a living cell culture is illustrated in
Figure 8 for events associated with apoptosis, a physicological process of cellular death resulting from an
intricate cascade of sequential interactions. Gene products directly involved in the chain of events can be
labeled by fusion to appropriate members of the fluorescent protein family (in this case, BFP and GFP) for
co-expression in the same cell in order to probe specific associations by FRET. The proteins involved with
apoptosis interact within the mitochondria and display a gradual decrease in binding as programmed cell
death proceeds. Thus, an image of donor emission (Figure 8(a)) contains only fluorescence from the BFP-
labeled proteins, while the corresponding acceptor emission profile (Figure 9(b)) illustrates signals due to
proteins labeled with GFP (and some contribution from donor emission). A FRET filter (Figure 8(c)), as
described below, reveals fluorescence derived from resonance energy transfer between the two proteins
Among the factors that may potentially affect the accuracy of fluorescence resonance energy transfer
measurements in general, several are highly specific to the optical microscope. A primary target in
microscopy investigations is to obtain high resolution images, and this requires particular attention to the
quality and performance of optical filters employed to spectrally discriminate among the absorption and
emission wavelengths of the donor and acceptor. In order to maximize the signal-to-noise ratio (without
deleteriously affecting the specimen or the process being investigated), it is necessary to carefully balance
the intensity and time of exposure to excitation light with the concentration of donor and acceptor
fluorophores and the detector efficiency. If the concentration of donor-acceptor fluorophores is excessive,
self-quenching can occur, affecting the accuracy of FRET measurements. Photobleaching is a problem
with all fluorophores, and can affect the donor-acceptor ratio, altering fluorescence measurements. Excess
illumination intensity can also damage specimens, particularly those containing living cells or tissues.
A technique known as donor photobleaching fluorescence resonance energy transfer (pbFRET),
which exploits the photobleaching process to measure FRET, is often applied in the study of fixed
specimens. Based on pixel-by-pixel analysis, the method has been applied to measure proximity
relationships between cell surface proteins labeled with fluorophore-conjugated monoclonal antibodies.
Photobleaching FRET is founded on the theory that a fluorophore is sensitive to photodamage only when
it is elevated to an excited state. Statistically, only a small proportion of molecules are in an excited state at
any one time, and therefore, fluorophores with longer fluorescence lifetimes have a higher probability of
suffering photodamage and exhibit a higher rate of photobleaching.
Experimental evidence supporting this concept has demonstrated that the photobleaching time of a
fluorophore varies inversely with its excited-state lifetime. The occurrence of resonance energy transfer
reduces the fluorescence lifetime of the donor molecule, effectively protecting it against photobleaching.
Calculations of pbFRET are based on the decreased rate of donor photobleaching relative to that
measured for the donor in the absence of resonance energy transfer. The measurement of
photobleaching in FRET studies requires a relatively long timeframe, and therefore is most applicable to
fixed cell specimens in which temporal data is not important and the effect on cell function from
photobleaching is not an issue. In some respects, the donor photobleaching technique is less complicated
than sensitized emission measurement, although fitting of time constants to photobleaching curves
involving multiple components presents some additional difficulties.
The energy transfer efficiency can also be determined by acceptor photobleaching techniques, in
which the change in donor emission quenching is measured by comparing the value before and after
selectively photobleaching the acceptor molecule. Analysis of the change in donor fluorescence intensity
in the same specimen regions, before and after removal of the acceptor, has the advantage of requiring
only a single specimen preparation, and directly relates the energy transfer efficiency to both donor and
acceptor fluorescence.
9/30/2014 Olympus Microscopy Resource Center | Fluorescence Resonance Energy Transfer (FRET) Microscopy - Introductory Concepts 10/12
Accurate measurement of fluorescence resonance energy transfer in the microscope requires
compensation for all of the potential error sources. A straightforward technique to correct for detection of
donor fluorescence with the acceptor emission filter and acceptor fluorescence with the donor emission
filter (due to crossover or spectral bleed-through) has been developed. The method also corrects for the
dependence of FRET on the concentrations of the donor and acceptor fluorophores. The measurement
strategy, which requires a minimum of spectral information, utilizes a combination of three filter sets and
can be readily implemented. The donor, FRET, and acceptor filter sets are designed to isolate and
maximize three specific signals: donor fluorescence, acceptor fluorescence attributable to FRET, and the
directly excited acceptor fluorescence, respectively. In practice, three different specimens, containing just
donor, just acceptor, and both donor and acceptor are examined with each of the three filter sets, and the
resulting data manipulated arithmetically to correct for crossover and for uncontrolled variations in donor-
acceptor concentrations.
Presented in Figure 9 are schematic illustrations of crossover (spectral bleed-through) and filter crosstalk,
two significant problems that must be overcome in order to achieve quantitative results in fluorescence
resonance energy transfer experiments. Crossover or bleed-through is manifested by an overlap of the
donor fluorescence emission spectrum with the bandpass region of the acceptor emission interference
filter in Figure 9, resulting in donor emission signal (unwanted wavelengths) being transmitted through the
emission filter. In contrast, filter crosstalk describes the minimum attenuation (blocking) level over a
specific range of two filters placed together in series, and is of concern when matching excitation and
emission filters for fluorescence sets. Dichromatic mirrors are often included in crosstalk evaluation of
fluorescence filter combinations. Although two emission filters are rarely placed in the light path at the
same time, the spectra are drawn together in Figure 9 to simultaneously illustrate both concepts. Note that
the two filter spectra (blue and red curves) represent light transmittance by the interference filters,
whereas the donor emission curve (green) is a plot of intensity versus wavelength.
Additional factors, which can potentially introduce significant errors, also require correction when steady
state FRET measurement techniques are employed. Furthermore, careful control of the donor and
acceptor fluorophore concentrations is desirable. Fluorophore concentration determinations can be
partially avoided through the application of time-resolved fluorescence measurements, which provide a
method of obtaining average lifetimes without a precise knowledge of donor concentrations. The technique
enables quantitative determination of donor-acceptor separation distances, and is based on
measurements of the donor lifetime in the presence and absence of the acceptor. Measuring fluorescence
intensity decay as a function of time elucidates the emission dynamics of the excited-state molecule, and
consequently, more detailed information about the nature of the donor-acceptor interaction may be
obtained. Graphical plots of intensity decay illustrate time-averaged details of the fluorescence decay
process (see Figure 10(a)), which are unresolved when employing steady state techniques.
Measurements indicating the same value for average lifetime, when recorded as steady state intensity
normalized to absorption, may correspond to significantly different decay curve shapes in time-resolved
data plots, indicating differences in the intermolecular processes involved.
The fluorescence lifetime () of a fluorophore is the characteristic time that a molecule exists in the excited
state prior to returning to the ground state. Representing fluorescence decay in a simplified single
exponential form following a brief pulse of excitation light, the fluorescence intensity as a function of time
(t) is given by the equation:
I(t) = I
exp (-t/)
where I(0) is the initial fluorescence emission intensity immediately after the excitation light pulse, and I(t)
is the fluorescence intensity measured at time t. The fluorescence lifetime () is defined as the time
required for the intensity to decay to 1/e of its initial value (approximately 37 percent of I(0); Figure 10(a)),
and is the reciprocal of the rate constant for fluorescence decay from the excited state to the ground state.
The primary overall advantage of time-resolved versus steady state FRET measurements is that donor-
acceptor separation distances can be mapped with greater quantitative accuracy. This results in part
because fluorescent lifetimes do not depend upon local intensity or concentration, and are largely
unaffected by photobleaching of the fluorophores. Fluorescence lifetimes are, however, highly sensitive to
fluorophore environment, and even molecules with similar spectra may display distinct lifetimes under
different environmental conditions. Because scattering does not affect fluorophore lifetimes,
measurements of lifetime variation can provide information that is specifically related to local molecular
9/30/2014 Olympus Microscopy Resource Center | Fluorescence Resonance Energy Transfer (FRET) Microscopy - Introductory Concepts 11/12
The lifetime of a fluorophore is subject to modification by numerous variables in the local
microenvironment, including factors such as hydrophobicity, oxygen concentration, ionic strength of other
media components, binding to macromolecules, and the proximity to acceptor molecules that can deplete
the excited state by resonance energy transfer. It is a significant practical advantage that lifetime
measurements can serve as absolute indicators of molecular interactions, and are independent of
fluorophore concentration.
Two general techniques commonly employed to measure fluorescent lifetimes are categorized as the
time-domain (pulsed, see Figure 10(a)) and frequency-domain (also termed phase-resolved; Figure
10(b)) methods. Time-domain lifetime measurements employ pulsed excitation light sources, and the
fluorescent lifetime is obtained by directly measuring the emission signal or by photon-counting detection.
The frequency-domain approach utilizes sinusoidal modulation of the excitation light source (obtained from
pulsed or modulated laser systems), and lifetimes are determined from the phase shift and demodulation
depth of the fluorescence emission signal. Each of these approaches to fluorescence lifetime imaging has
specific advantages and disadvantages, and both have been widely applied to conventional widefield,
confocal, and multiphoton microscopy.
Illustrated in Figure 10 are schematic diagrams representing the time domain and frequency domain
techniques for determination of fluorescence lifetimes. In the time domain approach (Figure 10(a)), the
specimen is excited by a brief pulse of laser light having a duration much shorter than the lifetime of the
excited species, and the exponential decay profile is measured as a function of time. Fluorescence decay
is usually a monoexponential function for a single fluorophore, but can display far more complex character
if the excited state has numerous relaxation pathways available in the environment. Sinusoidally modulated
light from a continuous wave laser coupled to an acousto-optical modulator is employed to excite the
fluorophore in frequency domain experiments (Figure 10(b)). The resulting fluorescence emission is
sinusoidally modulated at the same frequency as the excitation, but is accompanied by a phase shift and
reduction in the modulation depth. In the case of a single exponential decay, the fluorescence lifetime can
be calculated by determining either the degree of phase shift (), or the modulation ratio (M), using the
equations presented in Figure 10(b). If the two values are identical, fluorescence decay is truly composed
of a single exponential function. When more than one fluorescent species is present (or a single
fluorophore experiences a complex environment), the phase shift and modulation lifetimes should be
evaluated over a wide range of frequencies.
The time-domain technique for measuring fluorescence lifetime relies essentially on single-photon
counting and requires a detection system with sufficient temporal resolution to collect nearly 100 percent
of the photons generated by each excitation pulse. Although phase-resolved techniques are relatively less
demanding to perform, they are not generally as sensitive as the photon-counting approach. When phase
modulation is employed to resolve complicated multifluorophore lifetimes, long exposure times to damaging
excitation illumination can prove to be excessive for some specimens, and also may not provide sufficient
temporal resolution for live-cell processes. The preferred technique depends upon both the information
required from the investigation and the type of specimen being studied.
Fluorescence lifetime measurements have proven to be a sensitive indicator of FRET, and have particular
advantages in live-cell studies because of the independence of lifetime measurements upon factors such
as concentration and light path length, which are difficult to control in living specimens. A primary
advantage of performing FRET studies by fluorescence lifetime measurement lies in the fact that it is
possible to distinguish energy transfer even between donor-acceptor pairs with similar emission spectra.
When fluorescence lifetimes are measured directly (in contrast to the use of steady state values), a
determination of FRET is possible without the photodestruction of the donor or acceptor fluorophores.
Because FRET reduces the fluorescence lifetime of the donor molecule through energy transfer to the
acceptor, a direct comparison of the donor lifetime in the presence of the acceptor ((DA)) to that in the
absence of the acceptor ((D)), enables the calculation of a FRET efficiency value (E(T)) for each image
Depending upon the technique, fluorescence lifetime measurements require the specimen to be exposed
to either high-frequency repetitive pulses of excitation light, or to continuous sinusoidally modulated light.
In studies with living cells, the effect of high-intensity illumination must always be evaluated. Regardless of
the method, the reference lifetime of the donor without acceptor must be determined under experimental
9/30/2014 Olympus Microscopy Resource Center | Fluorescence Resonance Energy Transfer (FRET) Microscopy - Introductory Concepts 12/12
Contributing Authors
Brian Herman and Victoria E. Centonze Frohlich - Department of Cellular and Structural Biology, University of
Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229.
Joseph R. Lakowicz - Center f or Fluorescence Spectroscopy, Department of Biochemistry and Molecular
Biology, University of Maryland and University of Maryland Biotechnology Institute (UMBI), 725 West Lombard
Street, Baltimore, Maryland 21201.
Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac
Dr., The Florida State University, Tallahassee, Florida, 32310.
conditions identical to those of the donor-acceptor measurement. One means of accomplishing this with a
single specimen is to measure the donor-only lifetime after photobleaching destruction of the acceptor
following the energy transfer experiment.
In biological investigations, the most common applications of fluorescence resonance energy transfer are
the measurement of distances between two sites on a macromolecule (usually a protein or nucleic acid) or
the examination of in vivo interaction between biomolecular entities. Proteins can be labeled with synthetic
fluorochromes or immunofluorescent fluorophores to serve as the donor and acceptor, but advances in
fluorescent protein genetics now enable researchers to label specific target proteins with a variety of
biological fluorophores having differing spectral characteristics. In many cases, the amino acid tryptophan
is used as an intrinsic donor fluorophore, which can be coupled to any number of extrinsic probes serving
as an acceptor.
If macromolecules are labeled with a single donor and acceptor, and the distance between the two
fluorochromes is not altered during the donor excited state lifetime, then the distance between the probes
can be determined from the efficiency of energy transfer through steady state measurements, as
discussed above. In cases where the distance between the donor and acceptor fluctuates around a
distribution curve, such as protein assemblies, membranes, single-stranded nucleic acids, or unfolded
proteins (see the scenarios presented in Figure 11), FRET can still be employed to study the phenomena,
but time-resolved lifetime measurements are preferred. Several biological applications that fall into both
cases are illustrated in Figure 11, including conformational changes, dissociation or hydrolysis, fusion of
membrane-like lipid vesicles, and ligand-receptor interactions.
Although various methods are available for the measurement of fluorescence resonance energy transfer
in the optical microscope, none are completely without disadvantages. Some techniques require more
elaborate and expensive instrumentation, while others are based on assumptions that must be carefully
validated. Certain approaches are appropriate for fixed specimens, but cannot be applied to living cell
systems, while other methods must incorporate significant corrective calculations or data analysis
algorithms. It is certain, however, that FRET analysis shows great promise for further development in the
utility and scope of biological applications. Dramatic improvements in instrumentation have occurred in
recent years, particularly with respect to time-resolved techniques.
Fluorescence lifetime measurements that were only accomplished with extreme difficultly in the past are
now aided by mature picosecond and nanosecond technologies. Advances in fluorescent probe
development have produced smaller and more stable molecules with new mechanisms of attachment to
biological targets. Fluorophores have also been developed with a wide range of intrinsic excited state
lifetimes, and a significant effort is being placed on development of a greater diversity in genetic variations
of fluorescent proteins. Entirely new classes of fluorescent materials, many of which are smaller than
previous fluorophores and allow evaluation of molecular interactions at lower separation distances,
promise to improve the versatility of labeling and lead to new applications of the FRET technique.
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