Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology C:
Photochemistry Reviews
j our nal homepage: www. el sevi er . com/ l ocat e/ j phot ochemr ev
Review
Frster resonance energy transfer A spectroscopic nanoruler: Principle and
applications
Harekrushna Sahoo

Technische Universitt Dresden, Biotechnology Center, Tatzberg 47/49, 01307 Dresden, Germany
a r t i c l e i n f o
Article history:
Received 18 March 2011
Received in revised form27 April 2011
Accepted 2 May 2011
Available online 8 May 2011
Keywords:
Fluorescence resonance energy transfer
FRET methods
Quantumdots
Fluorophore labeling
Protein folding
a b s t r a c t
Frster resonance energy transfer (FRET) in association with the recent advancements in optical tech-
niques provides a way to understand the detailed mechanisms in different biological systems at the
molecular level. Improvements in wide-eld, confocal and two-photon microscopy facilitate the mea-
surements of two-dimensional spatial distribution in steady-state as well as dynamic bimolecular
interactions. In the recent decade, FRET became an exceptional uorescence-based technique due to
its potential advantages for studying the biological processes in living cells and more for spatial resolu-
tion at nanometer scale. In particular, FRET investigations have shown that biomolecules adopt different
conformational structures to perform their functions. In this review, the basic principles and applica-
tions of FRET in chemistry, biology, and physics are discussed. Along with, the recent improvements in
uorophore design and labeling and FRET measurement methods are briey mentioned.
2011 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2. Principle of FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3. Measuring techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1. Donor uorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2. Acceptor uorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.3. Spectral imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.4. Acceptor photobleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.5. Fluorescence anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4. FRET probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.1. Fluorophore labeling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.1. Material chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.2. Molecular sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.3. Polymer chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.4. Biomolecular interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.5. Folding dynamics and conformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.6. Hostpathology interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
5.7. Drug and ligand screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
5.8. Lipid membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
6. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Tel.: +49 0351 463 40326; fax: +49 0351 463 40342.
E-mail address: harekrushna.sahoo@biotec.tu-dresden.de
1389-5567/$20.00 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochemrev.2011.05.001
H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030 21
Harekrushna Sahoo received his MSc degree fromUtkal
University (India) in 2000. He nished his PhD at
Jacobs University Bremen (Germany) in 2007 on peptide
conformation and dynamics using uorescence-based
techniques with Prof. Werner M Nau. He started work-
ing on protein folding using uorescence as the primary
technique with Prof. Lila M Gierasch at University of
Massachusetts-Amherst (USA) in2007. In2010, he started
workingonextra-cellular matrixinthegroupof Prof. Petra
Schwille. His focus is to study chemical aspects of biolog-
ical processes.
1. Introduction
Frster resonance energy transfer (FRET), a well-established
photophysical phenomenonby whichenergy transfer froma donor
uorophore to an acceptor molecule (chromophore/uorophore)
occurs over long distances (typically from 1nm and up to 10nm)
as shown in Scheme 1, was rst established theoretically in 1948
[1]. FRET occurs through radiationless process (without emission
of photon), which can be better explained by a combination of
the quantumphysical model and the classical concept of Coulom-
bic dipoledipole interactions. The energy transfer efciency often
measures the relative distance between the donor and acceptor
(FRET pair) and therefore, is popularly known as molecular yard-
stick or ruler. Scheme 1 shows a typical FRET and No FRET
situation between donor and acceptor, where the donor and accep-
tors are conjugated to a short peptide chain(black and white circles
represents different amino acids).
FRET technique offers a fewpredominant advantages over cur-
rently usedother techniques inmany scientic researchareas, such
as molecular interactions as well as conformational and dynamic
changes in biomolecules. For instance, FRET is applied to investi-
gate the changes duringmolecular interactions as a functionof time
due to its noninvasive nature. This technique provides other advan-
tages, including increased sensitivity, short observation timescale
in nanosecond, the working range of distances over which most
of the biomolecular processes occur, the relative simplicity of the
experiment and the ability to apply it to dilute sample solutions
[25]. Consideringtheadvantages of FRET, it is particularlyeffective
in the detection of interacting membrane proteins for which assays
are limited with other traditional methods. Moreover, FRET assays
are adopted to monitor the dynamic processes of proteinprotein
interactions in vivo, such as intracellular signaling. The molecular
process-underlying FRET has been reviewed extensively [6,7].
FRET is combined with other uorescence spectroscopic meth-
ods to exploit the mechanisms of different biological processes
because of its non-interference characteristics. The combined
application of single-molecule uorescence method and FRET
in life sciences has taken a stronghold in recent years. In par-
ticular, single-molecule uorescence resonance energy transfer
has become a sensitive and powerful tool for determining con-
formational changes and molecular interactions [812]. Besides,
FRET also provides accurate measurements of inter- and intra-
molecular distances in free-diffusing as well as immobilized
biomolecules [8,10,11,1315]. FRET together with Fluorescence
Correlation Spectroscopy (FCS), is used for probing molecular
dynamics, kinetic, and photophysical properties, as illustrated in
several recent publications [11,1618]. Steady-state (wavelength
dependent)-FRET provides the average molecular information;
whereas time-resolved (time dependent)-FRET helps in resolving
the whole-distribution of molecules [19,20]. To accommodate a
longer measuring distance than 10nm(traditionally the optimum
distance for FRET), triple-FRET has been developed [21,22]. In case
of triple-FRET, three uorophores are used and thus, referred to
three-component energy transfer process. Triple-FRET involves the
rst energy transfer between a pair of uorophores and then the
acceptor fromthe rst energy transfer process serves as the donor
for the second energy transfer to the third uorophore or the sec-
ond acceptor. Recently, FRET with two-photon excitation system
has been applied in immuno assay development [23]. Basically,
two-photo excitation technique reduces the uorophore photo-
bleaching while diffusing through the observation volume.
Although, the photophysics and photochemistry of FRET is
well studied theoretically and experimentally for many years, it
only became applicable to biological/biochemical sciences after
the technical advancements in optical instrumentations and uo-
rophorechemistry[14,2429]. Furthermore, improvements inlight
microscopy imaging have generated a lot of interest obtaining
spatial and temporal distribution of protein associations in liv-
ing cells [30]. For example, combination of FRET with monoclonal
antibodies allowed to gain more insights into protein structures
in solutions, biological membranes and cell surface mapping of
molecules on immuno-competent cells [31]. FRET can also be used
in DNA sequencing and polymerase chain reactions [32]. In this
review, the principles and applications of FRET has been described
along with, brief discussions regarding the measurement methods
and uorophore-labeling techniques.
2. Principle of FRET
Jean Perrin (in 1920s) rst observed the excitation energy
transfer fromone molecule to another (separated within the non-
radiating near eld) through interactions between the oscillating
dipoles moments, which he termed as transfert dactivation.
Giving a correct theoretical basis to the energy transfer mech-
anism in 1948, Theodor Frster assumed that the oscillating
dipole moments are identical and the interaction energy is small
compared to the energies of the spectral transitions [33]. The prin-
ciple of FRET involves the radiationless energy transfer process
in which an energetically excited uorophore (donor) transfers
energy to another molecule (acceptor) through dipoledipole cou-
pling (through space). Furthermore, the excited acceptor molecule
returns to the ground state by losing its energy via photon emission
Scheme 1.
22 H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030
Fig. 1. Schematic representation of FRET demonstrating the energy transfer from excited donor (D*) to acceptor (A) via nonradiative process. Energy transfer excites the
acceptor fromA to A
*
followed by radiative energy relaxation in terms of uorescence. Solid and wavy arrows indicate the radiative and nonradiative processes, respectively.
Energy coupling between the ground states of donor and acceptor is shown in dotted lines.
(in case, acceptor is a uorophore), i.e., uorescence (Fig. 1). In the
case of FRET, the groundstate donor andthe acceptor molecules are
coupled energetically. The Jablonski diagram (Fig. 1) explains the
FRET process in a simplied way in terms of the donor/acceptor
excitation and emission and their energetically coupled ground
states. Irrespective of the photophysical characteristic of the accep-
tor, i.e., whether it is a chromophore or uorophore, the energy
transfer process is called as Frster resonance energy transfer.
Although, most widely used and IUPAC nomenclature is Frster res-
onance energy transfer, sometimes it is also referred as uorescence
resonance energy transfer.
Mechanistically, FRET is a two-step process that occur simulta-
neously as illustrated in Fig. 1, i.e. (i) excitation of photons from
the ground to excited state of the donor and energy transfer pro-
cess from excited donor to acceptor molecule through the dipolar
coupling between donor emission and acceptor excitation dipole
moments [25]. FRET efciency (E
FRET
) varies as the sixth power of
the distance between the two molecules (R) as shown in Fig. 2Aand
can be determined by the following equation:

FRET
=
R
6
0
R
6
0
+R
6
(1)
where R
0
, Frster radius or critical distance, is the characteristic
distance at a FRET efciency of 50%, which varies for different FRET
pairs. FRET efciency is close to maximumat distances less than R
0
,
and minimumfor distances greater than R
0
.
For distances close to R
0
, FRET is employed as a molecular ruler
due tohigher precisioninthe measurement anddata interpretation
[34,35]. R
0
in an aqueous solution is determined by a fairly simple
equation (Eq. (2)) with well-known input parameters:
R
0
= [8.79 10
5
(k
2
q
4
Q
D
j(z))]
1]6
(2)
where k
2
represents the angle between the two uorophore dipole
moments, Q
D
is the donor quantum yield and q is the refractive
index of the medium. J(z) is the spectral overlap integral between
the normalized donor uorescence, F
D
(z), and the acceptor absorp-
tion spectra (which is a direct measure of the molar extinction
coefcient,
A
(z)), as illustrated in Fig. 2B. J(z) is determined by
the following equation:
j(z) =

F
D
(z)
A
(z)z
4
dz

F
D
(z) dz
mol
1
cm
1
nm
4
(3)
E
FRET
has a sharp fall-off as shown in Fig. 2A with the increase in
FRET distance between FRET pair. E
FRET
is determined either from
the uorescence intensity (I
DA
: intensity of donor in presence of
acceptor and I
D
: intensity of donor only) or lifetime (z
DA
: lifetime
of donor in presence of acceptor and z
D
: lifetime of donor only) of
the donor molecule as follows,

FRET
= 1

l
DA
l
D

and
FRET
= 1

z
DA
z
D

(4)
Fig. 2. (A) Dependency of FRET efciency (EFRET) on the distance between FRET pair
(R). The dashed area in the curve (EFRET vs R) represents the sensitive FRET region
for the FRET pair. (B) Graphical representation of spectral overlapping, J(z), between
donor uorescence/emission spectra (blue) and acceptor absorption spectra (red).
H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030 23
There are some minor factors that affect FRET efciency, such
as, molecular brightness of uorophore, j(j=oQW(0), where
is the detection efciency of the uorescence emission, o is the
absorption cross-section at the excitation wavelength, Q is the u-
orescence quantum yield, and W(0) is the laser intensity at the
center of the point-spread function), stoichiometry ratio between
uorophores (which is the FRET pair labeling consequence; the
stoichiometry ratio between donor and acceptor that is outside
the range of 10:1 to 1:10) [36], and cross-talk or bleed-through
between uorophores (detection of donor signal in the acceptor
channel with donor excitation and vice versa). Fluorophores with
lowbrightness, interfere with the background noise and thus with
the measurement. To avoid this issue, the FRET pair must be of the
comparable brightness. Recently, Mller et al. in 2005 consolidated
Eq. (4) withbrightness-relatedparameters, whichis modiedtothe
following equation [18]

FRET
=
l
AD
l
DA
+l
AD
(5)
Here, I
AD
is the uorescence intensity of acceptor in the pres-
ence of a donor and is the detection-correction factor (=(
A,R
Q
A
/
D,G
Q
D
).
A,R
and
D,G
are the detection efciencies of accep-
tor and donor in red and green channels, respectively and Q
A
is the
uorescence quantumyield of acceptor.
Cross-talk between the uorophores, which complicates the
FRET interpretation, can be avoided by selecting uorophores with
larger separation in their emission spectra or employing other
methods such as ALEX (Alternating Laser Excitation) or PIE (Pulsed
Interleaved Excitation) [18]. Simultaneously, ALEX/PIE can also be
used to determine the labeling stoichiometry in FRET as well as the
accuracy of measurements by analyzing samples containing only
an active donor and acceptor molecules.
The FRET efciency depends on a number of factors; the most
integral criteria are described as follows:
(i) Spectral overlap integral, J(z): donor emission spectrum must
signicantly overlap with the absorption spectrum of the
acceptor.
(ii) Distance between FRET pairs, R: distance between the donor
and acceptor uorophores must fall within the range of 1 to
10nm. TheFRETefciencyis inverselyproportional tothesixth
power of R. Hence, a slight modication in R can signicantly
affect the FRET signals.
(iii) Dipoledipole interaction, k
2
: donor emission dipole moment,
the acceptor absorption dipole moment, and their separation
vectors must be in favorable mutual orientation. k
2
depends
on the angle between the dipole moments of the donor and
acceptor in much the same way as the position of a radio
antenna can affect its reception. If the donor and acceptor are
alignedparallel toeachother, the FRETefciency will be higher
than if they are perpendicular to one another. The degree
of alignment denes the value of k
2
, which varies between
0 (dipole moments are perpendicular to each other) and 4
(dipole moments parallel to each other). It is usually assumed
tobe2/3, whichis theaveragevalueintegratedover all possible
angles for freely rotating attached-uorophores.
3. Measuring techniques
Numerous methods have been used to measure FRET, such
as, change in donor uorescence or acceptor emission [37,38].
Although, it is not possible to discuss all of these methods here,
a few that are used more often, simple and easy to handle are
explained. Most of the methods mentioned here accommodate
the measurement of both types of FRET processes, i.e. hetero-FRET
(FRET betweentwodifferent chromophores) andhomo-FRET (FRET
Fig. 3. Schematic diagramof aTCSPC(timecorrelatedsinglephotoncounting) setup.
Blue and red dotted lines represent the excited and emitted signals, respectively.
TAC shows the dependency of voltage as a function of time. PMT: photo-multiplier
tube; CFD: constant fractional discriminator; TAC: time-to-amplitude converter;
and MCA: multi-channel analyzer.
between same chromophores). FRET methods can be divided into
the following fundamental categories.
3.1. Donor uorescence
Monitoring changes in donor uorescence (either lifetime, i.e.,
time-resolved or intensity, i.e., steady-state) in the presence and
absence of acceptor are the most direct method used for the FRET
measurements. The uorescence lifetime can be determined with
a time-correlated single photon counting (TCSPC) system with
a higher accuracy. The advantage of TCSPC system is its higher
detectionefciency, higher signal-to-backgroundratio, andits sim-
plicity. Following schematic diagram(Fig. 3) describes the working
principle of TCSPC system.
The most common variants of this approach are FLIM-FRET
(a method that measures donors uorescent lifetime, i.e., time
spent by the uorophore at the excited state), based on uores-
cent lifetime imaging (FLIM) [39,40]; and acceptor bleaching (an
approach that measures the intensity of donor uorescence before
and after photobleaching acceptors), which is explained later (Sec-
tion 3.4) [41]. The uorescence lifetime is independent of the
intensity of the uorophores, and therefore it is possible to use
any cell for FLIMFRET experiments. Another advantage is that
only the lifetime of the donor uorophore has to be measured
and thus, the acceptor uorophore might have inefcient emis-
sion or may even quench the emission. Fig. 4 describes a typical
FLIMsetup, where a TCSPC module is coupled to the microscope to
record the uorescence lifetime and an EMCCDcamera for spectral
imaging.
Similarly, measuringtheuorescenceintensityof adonor before
and after photobleaching the acceptor is equivalent to measuring
the intensity of the donor in the presence and absence of accep-
tors. Bleaching of the acceptors produces an increase in the donors
emission if FRET is occurring. In several cases, it has been found
that the acceptor quenches the donor uorescence with a contact-
quenching mechanism(which can be corrected on the basis of the
van der Waals radius of the acceptor molecule) [19].
3.2. Acceptor uorescence
One of the most popular methods used for measuring FRET
involve monitoring acceptor emission upon donor excitation.
Commonly referred to as the three-cube method, this technique
24 H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030
Fig. 4. Schematic representation of Fluorescence Lifetime Imaging Microscopy
(FLIM). Blue and red dotted lines display the excitation and emission paths, respec-
tively. Solid red arrow guides the uorescence signal for visualization through
eyepiece. PD: photo-detector and EMCCD: electron multiplying charge coupled
device.
involves acquiring three different images using three uorescent
lter sets. Firstly, a lter set that excites the donor but measures
emission fromthe acceptor is used to generate a FRET image. Sec-
ondly, images obtained with lter sets that measure emission from
donors or acceptors when directly excited are then used to correct
the FRET images. Recently, variants of this method calibrated using
either FLIMFRETor acceptor-bleaching have beenimplementedto
measure actual FRET efciencies [42,43]. The intensity observed in
the corrected FRET image not only encodes information about the
amount of FRETinthe sample, but is alsoa functionof the amount of
sample present, the excitationintensity, the excitationwavelength,
and the instrumentation used (lters, objectives, detectors, and the
like). An additional advantage of the three-cube approach is its
speed of data acquisition(typically requiring a fewseconds as com-
pared to minutes for most of the other FRET imaging approaches).
This FRET method is applied for time-lapse or three-dimensional
FRET imaging in living cells.
3.3. Spectral imaging
Recently, a photon-efcient FRET method has been developed,
which monitors changes in the intensity of donors and acceptors
simultaneously using spectral imaging [44]. Information about the
parameters like abundance of donors and acceptors, as well as
about the FRET efciency are encoded in the emission spectra of
donor and acceptor. Earlier, theoretical approaches for measuring
FRET using spectral imaging have beendescribedandimplemented
using two-photon microscopy (a formof microscopy in which two
coincident infrared photons, each with only half of the energy
required to excite a particular uorophore, are used to excite a u-
orophore) [45]. An advantage of using spectral imaging to measure
FRET is that in addition to yielding FRET efciency, it also mea-
sures the abundance of donors and acceptors. Because two-photon
excitationis usuallyimplementedwithtunable lasers, wavelengths
that efciently excite both donors and acceptors can be selected.
This is important because the judicious selection of excitation
Fig. 5. Representation of FRET fromuorescence anisotropy measurements. Donor
emission as a result of no-FRET displays high polarization (A); where as, depolariza-
tion is due to the acceptor emission as a result of FRET (B). Blue arrow: excitation
polarization direction, Black arrow: donor excitation dipole moment, Green arrow:
donor emission dipole moment, and Red arrow: acceptor emission dipole moment.
During no-FRET, uorescence fromdirectly exciteddonor is emittedwitha polariza-
tionsimilar toexcitationcausinghighpolarization. As consequenceof FRET, acceptor
emission undergoes depolarization compared to the donor excitation because of its
different orientation in the original photoselection plane.
wavelengths for specic samples can maximize the signal-to-noise
ratio of both donor and acceptor signals [46]. This FRET method
requires specialized equipment for spectral imaging, two-photon
excitation, and puried samples of the donor and acceptor uo-
rophores to provide reference spectra.
3.4. Acceptor photobleaching
Acceptor photobleaching method employs selective photo-
chemical destruction of the acceptor uorophore, which, if the
two uorophores had previously been physically close enough
to give FRET, results in a release from donor quenching and an
increase in donor emission [47,48]. Technically, the advantage of
this method is that it reduces the requirements for compensa-
tion and calibration associated with standard FRET and can be
performed on normal wide-eld microscopes. For accurate FRET
measurements, this technique requires complete bleaching of the
acceptor (which can take several minutes) without bleaching the
donor.
3.5. Fluorescence anisotropy
Fluorescence anisotropy can be used to measure the energy
transfer efciency between the FRET pair directly fromthe change
in anisotropy [7]. When a randomly oriented population of u-
orophores is excited with a linearly polarized light (excitation
polarization), molecules whose absorption/excitation dipoles are
oriented parallel to the polarization axis are preferentially excited
(Fig. 5A). The resulting anisotropy (r), a measure of the degree
of orientation, is determined by measuring the emission intensity
H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030 25
through vertically (I
V
) and horizontally (I
H
) oriented polarizers (Eq.
(6)).
r =
l
V
l
H
l
V
+2l
H
(6)
Depolarization occurs as a result of the emission from the
acceptor during FRET, i.e., when the donor transfers its excita-
tion energy to a neighboring and proximal acceptor molecule with
a different orientation compared to the original photoexcitation
plane (Fig. 5B). Particularly for uorescent proteins, depolarization
due to FRET can be distinguished from that of molecular rotation
as energy transfer can occur much more rapidly than molecu-
lar rotation. By knowing the uorescence quantum yield of the
donor and acceptor (
D
and
A
: uorescence quantum yield for
donor and acceptor, respectively) and the anisotropies (r
D
: emis-
sion anisotropy of donor and r
A
: emission anisotropy of acceptor
when excited through energy transfer), the total anisotropy (r
tot
)
of the donoracceptor pair cane be calculated with the following
equation [49],
r
tot
= r
D

tot
+r
A

tot

(7)
where
tot
is the combined quantum yield of donor and acceptor
(in case of homo-FRET, it is the same molecule). On substituting the
values of uorescence quantum yield with rate of energy transfer
[49], Eq. (7) can be modied as follows;
r
tot
= r
D
1 +(R
0
]R)
6
1 +2(R
0
]R)
6
+r
A
(R
0
]R)
6
1 +2(R
0
]R)
6
(8)
Substituting Eqs. (1) and (8) becomes as follows;
r
tot
= r
D

FRET
2
FRET
1
+r
A

FRET
1
2
FRET
1
(9)
In principle, Eqs. (7)(9) can be used together to estimate the
FRET efciency andFRET distance fromthe uorescence anisotropy
measurements.
Time-resolved uorescence anisotropy decay and steady-state
uorescence anisotropy are used to measure homo-FRET. Steady-
state intensity or lifetime uorescence (as mentioned above) is not
suitable for homo-FRET measurements as in homo-FRET, intensity
and lifetime values do not change (FRET between two identical
molecules in bidirectional, which leads to no resultant change
in uorescence intensity or lifetime). In details, energy transfer
between identical chromophores located at close enough distances
is possible, provided that there is signicant overlap of the absorp-
tion and emission spectra, which is the case for most uorescent
proteins including eGFP. The critical Frster distance for eGFPs is
about 4.7nm[50]. Quantitative analysis of uorescence anisotropy
decays provides information on structural parameters: distance
between the two interacting chromophores and spatial orienta-
tion between the chromophores within dimeric proteins. As far
as in vivo system is concerned, uorescence anisotropy decay is
not easy to measure under the microscope and the instrumenta-
tions are necessarily sophisticated. Recently it has been shown that
two-photon excitation steady-state FAIM(uorescence anisotropy
imaging microscopy) can be used easily for time-lapse homo-FRET
[26].
Single molecule uorescence technique can be coupled with
the above-discussed techniques to measure FRET of individual
donoracceptor pairs. There are several advantages with single
molecule over ensemble measurements. For example, inanensem-
ble measurement the properties of the individual molecules are
always get averagedout; whereas singlemoleculetechniquebuilds
up a distribution histogram of individual molecules as a func-
tion of time. Also, with its small excitation volume, it reduces the
Fig. 6. Schematic diagramof single molecule uorescence setup. Dotted green and
blue lines represent the excitation light with different wavelengths; where as red
dotted line is the emission path. As shown in the gure, the emission light is col-
lected in the same excitation back-focal plane but then using a beam splitter (or,
dichroic mirror), it is reected towards the detectors (APDor EMCCDcamera). APDs
(avalanche photo diode) are used as detectors to have the uorescence signal.
background noise mainly due to the Raman and Rayleigh scat-
tering, impurities, glass coverslips, optical components, etc. Fig. 6
explains the general single molecule setup coupled with a confocal
microscopy. Inthis setup, the emissionfromthe sample is collected
with the same excited plane but then reected with the beamsplit-
ter (or dichroic mirror) towards the detectors (APD). Before the
detectors, a ip mirror is used to divert the emission path towards
a camera (i.e., EMCCD camera) to collect the individual donor and
acceptor images.
4. FRET probes
Optimizing FRET pairs and their attachment to the biomolecules
are the two most-important parameters that can impact the accu-
racy of the data interpretation. Fluorophores can be typically and
broadly classied into two different types: intrinsic and extrinsic.
Intrinsic uorophores occur in nature and include aromatic amino
acids (tryptophan, tyrosine, andphenyl alanine), nicotinamide ade-
nine dinucleotide (NADH), avins, and derivatives of pyridoxal
and chlorophyll. Most of the intrinsic uorophores require exci-
tation by short wavelength (ultraviolet or blue light), which is
often hazardous to live cells. Additionally, other photophysical
properties such as, brightness and uorescence quantumyield are
generally lower for most of the practical applications of intrinsic
uorophores. These properties made the way for the develop-
ment of extrinsic uorophores, which are typically synthesized
from polyaromatic compounds having a conjugated -electron
system. Extrinsic uorophores are readily attached to the target
biomolecules in vitro systems, albeit have disadvantages in vivo
labeling. However, introduction of labeled molecules into cells has
been made possible by utilizing more invasive techniques [51].
26 H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030
Table 1
In vitro and in vivo labeling techniques showing the functional and reacting groups.
Labeling techniques Target group Labeling group Enzyme Ref.
Chemical Amine Succinimidyl ester [53]
Sulfonyl chloride [54]
Isothiocyanate [52]
Thiol Maleimide [55]
Iodoacetamide [56]
Alkyl halide [56]
Enzymatic Primary amine Amide Transglutaminase [60]
Carboxylic acid Amine Sortase [61]
Alcohol Nitrophenyl phosphonate Cutinase [62]
Thioester Thiol Intein [63]
Tagged Histidine Nickel-complex [64]
Aspartate Zinc-complex [65]
Lanthanide Terbium-complex [66]
Tetracysteine
a
Biarsenic [67]
CLIP-Tag Benzylcytosine [57]
Halo-Tag Aliphatic chloride [58]
SNAP-Tag Benzylguanine [59]
ybbr-Tag Serine sfp-phosphopantetheinyl transferase [68]
Genetical Fluorescent proteins
b
GFP [69]
CFP [70]
YFP [71]
RFP [72]
a
Tetracysteine tag is mostly used for attaching FlAsH/ReAsH, although it can be used for other uorophores as well.
b
GFP: Green Fluorescent Protein; CFP: Cyan Fluorescent Protein; YFP: YellowFluorescent Protein; and RFP: Red Fluorescent Protein.
4.1. Fluorophore labeling methods
Fluorophore labeling is one of the parameters, which can
adjudge the FRET data accuracy. Reactive uorescent dyes are
widely used to modify amino acids, peptides, proteins (in particu-
lar antibodies), oligonucleotides, nucleic acids, carbohydrates, and
other biological molecules. There are different techniques available
for labeling biopolymers. Among the reactive dyes, amine-reactive
dyes are most often used to prepare various conjugates for
immunochemistry, histochemistry, uorescence in situ hybridiza-
tion (FISH), cell tracing, receptor binding, and other biological
applications as amino groups are either abundant or easily intro-
duced into biomolecules [5254]. In general, thio-reactive reagents
are frequently used to develop probes for investigating some par-
ticular protein structure and functions [55,56]. In chemical biology,
several tagged-methods are used to attach the uorophores to the
target biomolecules, i.e. SNAP, CLIP, Halo (see Table 1) [5759].
Lastly, the preferred uorophores should have high uorescence
quantumyields and retain the biological activities upon the attach-
ment to the biomolecules.
There are several methods for uorophore attachment to the
target molecules. BelowinTable 1briey describes commonly used
methods.
Depending upon experimental requirements and existing label-
ing techniques (described in Table 1), in vitro labeling can be
performed more readily than that of in vivo. Particularly in the
case of uorescent protein variants (i.e., Green Fluorescent Protein,
GFP), the probes are large enoughcomparedto the movements that
they report, and quite exibly attached to the proteins of inter-
est allowing for wide differences between the distances of the two
probes and their attachment points. Additionally, uorescent pro-
tein labeling can induce artifacts via homo-dimerization [73].
Commonly used uorophores (classied into two different
classes, e.g., ultra-violet andvisibleuorophores) as FRETpairs with
their characteristic Frster distances are illustrated in Table 2.
A new group of FRET probes, i.e. nanocrystal (quantum dots:
QDs), have been employed for nearly a decade and can act as
either donors or acceptors [77,78]. Most of the QDs used in biolog-
ical applications are synthesized from CdSe (Cadmium Selenide)
cores overcoated with a layer of ZnS (Zinc Sulde). Quantum dots
in particular exhibit critical properties of the uorophores (such
as high quantum yield and extinction coefcient, brightness, and
photostability). Given the multiplexing capabilities [7981] and
photostability, QDs exhibit an additional feature of great utility:
the number of groups bound to their surface can be varied from
1 to few 10s in a controlled manner. Another striking advan-
Table 2
Forster distances of different FRET pairs (ranging fromultra-violet to visible).
Ultra-violet FRET pairs Visible FRET pairs
FRET pair (D/A) R
0
/() Ref. FRET pair (D/A) R
0
/() Ref.
Tryptophan (Trp)/DBO 9 [19] Alexa 488/Alexa 594 54 [69]
Tryptophan/Dansyl Chloride 21 [70] Alexa 488/Alexa 647 39 [71]
Naphthalene/Dansyl Chloride 27 [72] Alexa 555/Alexa 647 51 [73]
EDANS/Dabcyl 33 [74] ATTO 550/ATTO 647 65 [75]
ATTO 390/ATTO 425 41
a
Cy3/Cy5 60 [75]
GFP
3
/YFP 56 [76]
GFP/RFP 47 [76]
CFP/YFP 49 [76]
a
R
0
value obtained from www.atto-tec.com. The above R
0
values consider the attachment of the FRET pair to the target molecule and thus, it might vary when the
uorophores are not attached to the target molecule.
H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030 27
tage is their emission wavelength, which is a function of their
sizes [80]. Their use as probes in living cells is rapidly increas-
ing due to the advantages and compatibility (and some extent the
non-toxicity) with in living systems. As a result of their extreme
brightness andcomparatively long lifetime, QDs are easily detected
and identied visually and by electronic imaging providing single
molecule sensitivity, positional super-resolution [82] and conr-
mation of molecular identity (e.g. of substances to which they are
conjugated). As FRET donors, QDs benet fromtheir large absorp-
tion cross-section that increases continuously from their narrow
emission bands to the ultra-violet. Thus, despite their relatively
large size (which can be considered as one of the drawbacks)
and the requirement proximal acceptor(s) to the nanoparticle
surface, QDs function efciently as FRET donors in many bio-
analytical and imaging applications [37,83,84]. And, because of
the range of the absorbance peak for QDs, which ranges from
ultra-violet to violet, these are mostly used as FRET donors.
Development in QDs with smaller stabilizationconjugation coats
might enable FRET probing at locations removed fromthe particle
surface.
5. Applications
FRET has been utilized as a Spectroscopic Nano-Ruler to
monitor the structural and conformational perturbations in nano
and sub-nano scales [35]. In biological science, conformational
changes are somewhat more demanding in order to understand
the underlying bioprocess mechanisms. In recent times, FRET has
frequently been employed to measure conformations and related
transitions of the molecule at the molecular level [8,10,11,85].
It is noteworthy that the FRET efciency depends not only on
the distance of the two uorophores, but also on their relative
orientation, i.e., their absorption and emission dipole moments.
However, latter effect is however usually suppressed because
it would be necessary to rigidly attach the donor and accep-
tor to the molecule of interest. Usually, exible linkers are used
for uorophore labeling, thereby averaging over many possi-
ble orientations, thus limiting the observable distance range to
a few nanometers. FRET is an useful biophysical tool to quan-
tify molecular dynamics, including proteinprotein interactions,
proteinDNA interactions, and protein conformational changes
both in vitro and in vivo systems. Scientic research elds in which
FRET has been applied substantially and distinctly are described
below.
5.1. Material chemistry
Fluorescence resonance energy transfer (FRET) has beenapplied
tocell-material interface for bothtwoandthree-dimensional adhe-
sion substrates to quantitatively analyze parameters [86]. FRET is
being utilized to quantify several parameters of the cellmaterial
interface relevant to cell response, including molecular changes
in matrix proteins induced by interactions both with surfaces and
cells. The mechanismof cellmaterial interactions at the molecular
level and the quantication of the cell-based nanoscale rearrange-
ment in the material has been investigated by using FRET [87].
Such techniques allow both dynamic and 3D analyses that are
useful to quantitatively relate downstreamcellular responses (e.g.
gene expression) to the composition of this interface. FRET has
also been implemented in coreshell nanoparticles complex sys-
tems, which offer different environments for probes associated
with them by different means. Using pyrene and coumarine as a
FRET pair, the compartmentalization of the core-shell nanoparticle
has been investigated [88].
5.2. Molecular sensor
FRET has been applied as sensor in various research areas due
to its advantages in investigating the static and dynamic states of
a macromolecule. Recently, different variants of coumarin dye are
used as FRET pairs to study the pH regions for sulfadimethoxine
and sulfamethizole [89]. Furthermore, the compartmentalized sig-
naling of kinase and second-messenger dynamics and the unique
features of genetic abilities with targeting and encoding are
unveiled with FRET-based biosensors that allowa real-time track-
ing of activity dynamics with high spatiotemporal resolution [90].
Where the conventional biochemical processes were unable to
uncover the spatio-temporal activity of a small GTPase in live cells,
it was possible to investigate by FRET-based biosensors [91].
5.3. Polymer chemistry
Numerous FRET studies have been done with polymers of var-
ious small molecules as a spacer between the FRET pairs. Such as,
polyphenylene dendrimers [92], amino acids [19,35,9395], carbo-
hydrates [96], and biomolecules [97] are used as spacer between
the donor and acceptor to reveal there rigidity depending on chain
lengthand also different uorophores are tried withthe same set of
spacers to have more accuracy with the FRET distances [94,74]. Fol-
lowing gure (Fig. 7B) displays the FRET dependency on the length
of a peptide chains. In this case, glycine-serine (Gly-Ser) units are
used as the separation between the FRET pair (Trp and DBO) as
shown in Fig. 7A [19].
Studyof biopolymers of different chainlengths withaminoacids
as the spacer provides an idea about the exibility at different
regions in a three-dimensional protein structure. To understand
the detailed role of carbohydrates on bioprocesses, FRET has been
used to investigate the conformational changes in carbohydrates
by labeling the uorophore through biotin-streptavidin chemistry
[96].
5.4. Biomolecular interactions
FRET and FLIM have been applied extensively to biomolecular
interaction studies: in the analysis of proteinprotein interac-
tions with high spatial and temporal specicity (e.g. clustering),
in the study of conformational changes, in the analysis of binding
sequences, and in applications such as high-throughput screen-
ing [98100]. Recent advances in microscopy have allowed the
development of new methods to analyze proteinprotein interac-
tions at very high resolution in both xed and live cells [101103].
Additionally, molecular interactions and conformational changes
of various proteins involved in the regulation of cell adhesion
and motility have been investigated [104]. FRET measurement of
Secretin docking with its receptor provides strong evidence for the
orientation of peptide-binding and signaling domains of a proto-
typic Class II G protein-coupled receptor [105].
5.5. Folding dynamics and conformations
FRET has beenemployedtodetermine the biomolecular dynam-
ics and conformations occurring in biological pathways. For
example, RNA folding [106], small DNA bending angles, the func-
tional meaning of which may be just as important as of large
bends (DNA bending) and DNAprotein interactions [107]. Fig. 8
displays a general scheme that shows the extractionof proteinfold-
ing intermediates and their dynamic information fromthe distance
distributions through FRET analysis. Basically, based on the FRET
distances between different sites (of interest) in a protein, charac-
terization of different intermediate states can be carried out. The
distance distribution probabilities in different intermediates reveal
28 H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030
Fig. 7. Fluorescence intensity of donor as a function of peptide chain length; (A)
structural representation of the peptide chain as a function of Gly-Ser units (where
n =0, 1, 2, 6, and10). Where, Trp(at N-termini) andDBO(at C-termini) serve as donor
and acceptor, respectively (taken fromJ. Phys. Chem. B 111 (2007) 26392646). (B)
Decrease in donor uorescence intensities, which alternatively indicate the FRET
efciency, as a functionof number of interveningaminoacids (n). Reference peptide:
Trp-(Gly-Ser)
6
COOH.
The spectra are adapted fromRef. [19].
the compactness of the protein and consequently the conforma-
tional changes during the folding process.
Recently, single molecule FRET (sm-FRET) of different confor-
mations of the tumor suppressor gene (p53) study shows that the
Fig. 8. Protein folding funnel. FRET analysis showing different energy-related inter-
mediates during protein folding pathway. The unfolded states are comparatively
more exible than the native 3-dimensional tertiary state. Unfolded states with dif-
ferent energies folds to a rigid and native state through several intermediate states
that differs in their energy distributions.
The folding funnel is taken fromRef. [9].
N-terminal domain weakly binds to the DNAbinding domain of the
p53. Together with time-resolved FRET (tr-FRET), different confor-
mations of the different domains in p53 were monitored, which are
mostly overlooked in ensemble FRET technique [13]. FRET analy-
ses revealed that the interaction between Fibronectin domains (III
Aand III B) exposes the binding sites in solution through conforma-
tional changes in Fibronectin III [108]. Besides the conformational
studies, FRET in combination with single-molecule steered the pro-
tein folding research to a high level [109]. Earlier, Ha et al. [10]
demonstrated the single molecule uorescence, which was later
implemented in protein folding by Hochstrasser and coworkers
[110] and Weiss and coworkers [8]. A unique aspect of the free
diffusion experiments on single molecules is that structural and
dynamic properties of the subpopulationof unfoldedmolecules can
be separately investigated at equilibriumin the presence of a large
excess of folded molecules, where the ensemble averaged proper-
ties are dominated by the folded state. A consistent nding in all of
thefreediffusionexperiments is that theoverall sizeof theunfolded
protein, as obtainedfromtheFRETefciency-determined, increases
continuously with increasing in denaturant concentration. This
behavior, rst unequivocally demonstrated for cold-shock-protein
(CspTm), chymotrypsin inhibitor 2 (CI2) [8], cyl-CoA binding pro-
tein (ACBP) [111], RNase H [112], protein L [113], the B domain
of protein A [41], the immunity protein Im9 [15] and the prion-
determining domain of Sup35 [114].
5.6. Hostpathology interactions
Giventhe advantages andcompatibility of FRETwithother tech-
niques, its use in understanding hostpathogen interplay has, to
date, been surprisingly limited [115]. FLIMFRET facilitates the
use of FRET to measure the physical distance between donor
and acceptor uorophores, a technique utilized by Latz et al. to
decipher signaling associated with immune recognition of CpG
DNA, a pathogen-associated molecular pattern (PAMP), by the toll-
like receptor, TLR9 [116]. Fluorescence lifetime imaging is rapidly
becoming a preferred method for making FRET measurements.
This technique has been utilized to investigate plant cell inva-
sion by the ascomycete fungus [117]. FRET measurements also
allow the subcellular localization processes like, binding of SH2
(Src Homology 2) domain of Hck, a tyrosine-protein kinase, to
tyrosine-phosphorylated cytoplasmic domain of CEACAM3 (carci-
noembryonic antigen-related cell adhesion molecule 3) in intact
cells, during bacterial infection [118]. FRET-based assays are valu-
able tools to resolve bacteria-induced proteinprotein interactions
in the context of the intact host cell.
5.7. Drug and ligand screening
TR-FRET (time-resolved FRET) brings together the low back-
ground benets of TRF with the homogeneous assay format of
FRET. This powerful combination provides signicant benets
to drug discovery researchers including assay exibility, relia-
bility, increased assay sensitivity, higher throughput and fewer
false positive/false negative results. Compared to other binding
assays, the polarization [119] and HTRF (high-throughput TR-
FRET)[120] binding assays are nonradiaoactive, therefore safer to
perform, yet very sensitive and homogeneous, therefore easier
and faster to automate. These methods are thus suitable for ef-
cient drug high-throughput screening procedures and can easily be
applied to other G protein-coupled receptor models. FRET-based
assay are used to screen a G-protein-coupled receptor-focused
library of uorescent compounds on the human eGFP-tagged
apelin receptor [121]. A series of uorescent ligands designed for
vasopressin and oxytocin G protein-coupled receptors was syn-
thesized and characterized to develop uorescence polarization or
H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 2030 29
homogeneous time-resolved uorescence (HTRF) binding assays
[122].
5.8. Lipid membrane
Loura and co-workers have reviewed the application FRET in
detecting and characterizing the phase separation in lipid bilayers
(bothinmodel systems andincell membranes) [123]. This reviewis
alsofocusedonmodels describingtherateandefciencyof FRETfor
bothuniformprobe distributionandphase separation, andrecently
reported methods for detection of membrane heterogeneity and
determination of phase boundaries, probe partition coefcients
and domain size. FRET is also applied to characterize single-phase
lipid systems, gel/uid phase separation, liquid ordered/liquid dis-
ordered phase separation (lipid rafts), complex systems containing
ceramide andcell membranes are presentedtoillustrate the wealth
of information that can be inferred from carefully designed FRET
studies of membrane domains. The intermembrane contact areas
between single unilamellar vesicles and planar supported bilayers
are quantied using FRET as basic tool [124]. In a recent review,
quantication of interaction of membrane proteins with biologi-
cally relevant lipid molecules using FRET methodologies has been
focused in details [125]. In the eld of proteinlipid interactions,
FRET has also been extensively used to characterize and quan-
tify partition of membrane proteins to lipid membranes [126] and
particular lipid phases [127], and most notably to liquid-ordered
(raft-like) phases [128].
6. Conclusions and outlook
With the recent advancements in optical instrumentations,
FRET along with other existing optical techniques is used to reveal
dynamics and interactions of uorescently labeled molecules with
very high precision. Thus, FRET is becoming considerably impor-
tant in combination with single molecule uorescence microscopy
[8]. Because of the non-invasiveness, simple, robust, and design
of genetically encoded uorophores, FRET is of tremendous use in
life science research. FRET also enables the quantitative measure-
ment of molecular interactions inlivingcells andeveninorganisms.
In this review, the pitfall of FRET data interpretation in terms
of the uorophore selection and their attachment to the target
molecules has been discussed. Also the use of different excitation
schemes, whichcanbring the accuracy withregards to cross-talk, is
mentioned.
Major new FRET developments in the area of uorescence life-
time determinations, either in the time or frequency domain will
exploit the techniques like, FLIM (or FLI, uorescence lifetime
imaging), which can be combined with multispectral, polarization-
sensitive, and optical-sectioning modalities and as such offers the
prospect of a do-it-all form of uorescence microscopy [129].
Besides, combination of FRET with techniques like single-molecule
and FCS exposes the dynamic processes in biomolecular processes
[130]. Also, the emergence of numerous new techniques based
on parameter modulation and perturbation, permitting the reli-
able phase-sensitive detection of extremely low FRET signals are
anticipated in the years to come. Signicant driving technology in
the areas of illumination sources (LEDs, Laser Emitting Diodes),
advanced optical imaging techniques, and improvements in the
theoretical framework dictating modes of data (image) acquisi-
tion and analysis will make FRET a more powerful technique
in investigating the biological fundamental processes. Further-
more, efforts devoted to facile introduction of diverse organic
and nanoparticle probes into cells is expected to exploit and
establish mixed kind of FRET pair (organic and nanoparticle
probes).
Acknowledgments
Dr. Jan K. Rainey (Dalhousie University, Canada), Dr. Danilo
Roccatano (Jacobs University Bremen, Germany), and Dr. Fang
Huang (China University of Petroleum-Shandong, China) are highly
acknowledged for their critical reading and lending valuable sug-
gestions. Financial support by the German Research Foundation
(DFG) within the Transregio SFB 67 is gratefully acknowledged.
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