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Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 20– 30

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology C:Photochemistry Reviews

j o ur nal homep age : www.elsev ier .com/ locate / jphotochemrev

eview

örster resonance energy transfer – A spectroscopic nanoruler: Principle andpplications

arekrushna Sahoo ∗

echnische Universität Dresden, Biotechnology Center, Tatzberg 47/49, 01307 Dresden, Germany

r t i c l e i n f o

rticle history:eceived 18 March 2011eceived in revised form 27 April 2011ccepted 2 May 2011vailable online 8 May 2011

a b s t r a c t

Förster 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 themolecular level. Improvements in wide-field, confocal and two-photon microscopy facilitate the mea-surements of two-dimensional spatial distribution in steady-state as well as dynamic bimolecularinteractions. In the recent decade, FRET became an exceptional fluorescence-based technique due to

eywords:luorescence resonance energy transferRET methodsuantum dotsluorophore labelingrotein folding

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 differentconformational 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 influorophore design and labeling and FRET measurement methods are briefly mentioned.

© 2011 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212. Principle of FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213. Measuring techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.1. Donor fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.2. Acceptor fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.3. Spectral imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.4. Acceptor photobleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.5. Fluorescence anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

4. FRET probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254.1. Fluorophore labeling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.1. Material chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.2. Molecular sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.3. Polymer chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.4. Biomolecular interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.5. Folding dynamics and conformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275.6. Host–pathology interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285.7. Drug and ligand screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285.8. Lipid membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

6. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Tel.: +49 0351 463 40326; fax: +49 0351 463 40342.E-mail address: harekrushna.sahoo@biotec.tu-dresden.de

389-5567/$20.00 © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jphotochemrev.2011.05.001

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H. Sahoo / Journal of Photochemistry and Photo

Harekrushna Sahoo received his MSc degree from UtkalUniversity (India) in 2000. He finished his PhD atJacobs University Bremen (Germany) in 2007 on peptideconformation and dynamics using fluorescence-basedtechniques with Prof. Werner M Nau. He started work-ing on protein folding using fluorescence as the primarytechnique with Prof. Lila M Gierasch at University ofMassachusetts-Amherst (USA) in 2007. In 2010, he startedworking on extra-cellular matrix in the group of Prof. PetraSchwille. His focus is to study chemical aspects of biolog-ical processes.

. Introduction

Förster resonance energy transfer (FRET), a well-establishedhotophysical phenomenon by which energy transfer from a donoruorophore to an acceptor molecule (chromophore/fluorophore)ccurs over long distances (typically from 1 nm and up to 10 nm)s shown in Scheme 1, was first established theoretically in 19481]. FRET occurs through radiationless process (without emissionf photon), which can be better explained by a combination ofhe quantum physical model and the classical concept of Coulom-ic dipole–dipole interactions. The energy transfer efficiency ofteneasures the relative distance between the donor and acceptor

FRET pair) and therefore, is popularly known as “molecular yard-tick or ruler.” Scheme 1 shows a typical “FRET” and “No FRET”ituation between donor and acceptor, where the donor and accep-ors are conjugated to a short peptide chain (black and white circlesepresents different amino acids).

FRET technique offers a few predominant advantages over cur-ently used other techniques in many scientific research areas, suchs molecular interactions as well as conformational and dynamichanges in biomolecules. For instance, FRET is applied to investi-ate the changes during molecular interactions as a function of timeue to its noninvasive nature. This technique provides other advan-ages, including increased sensitivity, short observation timescalen nanosecond, the working range of distances over which mostf the biomolecular processes occur, the relative simplicity of thexperiment and the ability to apply it to dilute sample solutions2–5]. Considering the advantages of FRET, it is particularly effectiven the detection of interacting membrane proteins for which assaysre limited with other traditional methods. Moreover, FRET assaysre adopted to monitor the dynamic processes of protein–proteinnteractions in vivo, such as intracellular signaling. The molecularrocess-underlying FRET has been reviewed extensively [6,7].

FRET is combined with other fluorescence spectroscopic meth-ds to exploit the mechanisms of different biological processesecause of its non-interference characteristics. The combined

pplication of single-molecule fluorescence method and FRETn life sciences has taken a stronghold in recent years. In par-icular, single-molecule fluorescence resonance energy transferas become a sensitive and powerful tool for determining con-

Scheme

y C: Photochemistry Reviews 12 (2011) 20– 30 21

formational changes and molecular interactions [8–12]. Besides,FRET also provides accurate measurements of inter- and intra-molecular distances in free-diffusing as well as immobilizedbiomolecules [8,10,11,13–15]. FRET together with FluorescenceCorrelation Spectroscopy (FCS), is used for probing moleculardynamics, kinetic, and photophysical properties, as illustrated inseveral recent publications [11,16–18]. Steady-state (wavelengthdependent)-FRET provides the average molecular information;whereas time-resolved (time dependent)-FRET helps in resolvingthe whole-distribution of molecules [19,20]. To accommodate alonger measuring distance than 10 nm (traditionally the optimumdistance for FRET), triple-FRET has been developed [21,22]. In caseof triple-FRET, three fluorophores are used and thus, referred tothree-component energy transfer process. Triple-FRET involves thefirst energy transfer between a pair of fluorophores and then theacceptor from the first energy transfer process serves as the donorfor the second energy transfer to the third fluorophore or the sec-ond acceptor. Recently, FRET with two-photon excitation systemhas been applied in immuno assay development [23]. Basically,two-photo excitation technique reduces the fluorophore photo-bleaching while diffusing through the observation volume.

Although, the photophysics and photochemistry of FRET iswell studied theoretically and experimentally for many years, itonly became applicable to biological/biochemical sciences afterthe technical advancements in optical instrumentations and fluo-rophore chemistry [14,24–29]. Furthermore, improvements in lightmicroscopy imaging have generated a lot of interest obtainingspatial and temporal distribution of protein associations in liv-ing cells [30]. For example, combination of FRET with monoclonalantibodies allowed to gain more insights into protein structuresin solutions, biological membranes and cell surface mapping ofmolecules on immuno-competent cells [31]. FRET can also be usedin DNA sequencing and polymerase chain reactions [32]. In thisreview, the principles and applications of FRET has been describedalong with, brief discussions regarding the measurement methodsand fluorophore-labeling techniques.

2. Principle of FRET

Jean Perrin (in 1920s) first observed the excitation energytransfer from one molecule to another (separated within the non-radiating near field) through interactions between the oscillatingdipoles moments, which he termed as “transfert d’activation”.Giving a correct theoretical basis to the energy transfer mech-anism in 1948, Theodor Förster assumed that the oscillatingdipole moments are identical and the interaction energy is smallcompared to the energies of the spectral transitions [33]. The prin-ciple of FRET involves the radiationless energy transfer process

in which an energetically excited fluorophore (donor) transfersenergy to another molecule (acceptor) through dipole–dipole cou-pling (through space). Furthermore, the excited acceptor moleculereturns to the ground state by losing its energy via photon emission

1.

22 H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 20– 30

F excitea ce. SolE d line

(ccFesttAor

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wdpa

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wmittct

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of donor in presence of acceptor and �D: lifetime of donor only) ofthe donor molecule as follows,

EFRET = 1 −(IDA

ID

)and EFRET = 1 −

(�DA

�D

)(4)

ig. 1. Schematic representation of FRET demonstrating the energy transfer from

cceptor from A to A* followed by radiative energy relaxation in terms of fluorescennergy coupling between the ground states of donor and acceptor is shown in dotte

in case, acceptor is a fluorophore), i.e., fluorescence (Fig. 1). In thease of FRET, the ground state donor and the acceptor molecules areoupled energetically. The Jablonski diagram (Fig. 1) explains theRET process in a simplified way in terms of the donor/acceptorxcitation and emission and their energetically coupled groundtates. Irrespective of the photophysical characteristic of the accep-or, i.e., whether it is a chromophore or fluorophore, the energyransfer process is called as Förster resonance energy transfer.lthough, most widely used and IUPAC nomenclature is Förster res-nance energy transfer, sometimes it is also referred as fluorescenceesonance energy transfer.

Mechanistically, FRET is a two-step process that occur simulta-eously as illustrated in Fig. 1, i.e. (i) excitation of photons fromhe ground to excited state of the donor and energy transfer pro-ess from excited donor to acceptor molecule through the dipolaroupling between donor emission and acceptor excitation dipoleoments [25]. FRET efficiency (EFRET) varies as the sixth power of

he distance between the two molecules (R) as shown in Fig. 2A andan be determined by the following equation:

FRET = R60

R60 + R6

(1)

here R0, Förster radius or critical distance, is the characteristicistance at a FRET efficiency of 50%, which varies for different FRETairs. FRET efficiency is close to maximum at distances less than R0,nd minimum for distances greater than R0.

For distances close to R0, FRET is employed as a molecular rulerue to higher precision in the measurement and data interpretation34,35]. R0 in an aqueous solution is determined by a fairly simplequation (Eq. (2)) with well-known input parameters:

0 = [8.79 × 10−5(�2�−4QDJ(�))]1/6

Å (2)

here �2 represents the angle between the two fluorophore dipoleoments, QD is the donor quantum yield and � is the refractive

ndex of the medium. J(�) is the spectral overlap integral betweenhe normalized donor fluorescence, FD(�), and the acceptor absorp-ion spectra (which is a direct measure of the molar extinctionoefficient, εA(�)), as illustrated in Fig. 2B. J(�) is determined by

he following equation:

(�) =∫FD(�)εA(�)�4 d�∫

FD(�) d�mol−1 cm−1 nm4 (3)

d donor (D*) to acceptor (A) via nonradiative process. Energy transfer excites theid and wavy arrows indicate the radiative and nonradiative processes, respectively.s.

EFRET has a sharp fall-off as shown in Fig. 2A with the increase inFRET distance between FRET pair. EFRET is determined either fromthe fluorescence intensity (IDA: intensity of donor in presence ofacceptor and ID: intensity of donor only) or lifetime (�DA: lifetime

Fig. 2. (A) Dependency of FRET efficiency (EFRET) on the distance between FRET pair(R). The dashed area in the curve (EFRET vs R) represents the sensitive FRET regionfor the FRET pair. (B) Graphical representation of spectral overlapping, J(�), betweendonor fluorescence/emission spectra (blue) and acceptor absorption spectra (red).

biology C: Photochemistry Reviews 12 (2011) 20– 30 23

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3

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Fig. 3. Schematic diagram of a TCSPC (time correlated single photon counting) setup.Blue and red dotted lines represent the excited and emitted signals, respectively.

H. Sahoo / Journal of Photochemistry and Photo

There are some minor factors that affect FRET efficiency, suchs, molecular brightness of fluorophore, ( = ��QW(0), where �s the detection efficiency of the fluorescence emission, � is thebsorption cross-section at the excitation wavelength, Q is the flu-rescence quantum yield, and W(0) is the laser intensity at theenter of the point-spread function), stoichiometry ratio betweenuorophores (which is the FRET pair labeling consequence; thetoichiometry ratio between donor and acceptor that is outsidehe range of 10:1 to 1:10) [36], and cross-talk or bleed-throughetween fluorophores (detection of donor signal in the acceptorhannel with donor excitation and vice versa). Fluorophores withow brightness, interfere with the background noise and thus withhe measurement. To avoid this issue, the FRET pair must be of theomparable brightness. Recently, Müller et al. in 2005 consolidatedq. (4) with brightness-related parameters, which is modified to theollowing equation [18]

FRET = IAD

˛IDA + IAD(5)

Here, IAD is the fluorescence intensity of acceptor in the pres-nce of a donor and is the detection-correction factor ( = (�A,R

A/�D,G QD). �A,R and �D,G are the detection efficiencies of accep-or and donor in red and green channels, respectively and QA is theuorescence quantum yield of acceptor.

Cross-talk between the fluorophores, which complicates theRET interpretation, can be avoided by selecting fluorophores witharger separation in their emission spectra or employing other

ethods such as ALEX (Alternating Laser Excitation) or PIE (Pulsednterleaved Excitation) [18]. Simultaneously, ALEX/PIE can also besed to determine the labeling stoichiometry in FRET as well as theccuracy of measurements by analyzing samples containing onlyn active donor and acceptor molecules.

The FRET efficiency depends on a number of factors; the mostntegral criteria are described as follows:

(i) Spectral overlap integral, J(�): donor emission spectrum mustsignificantly overlap with the absorption spectrum of theacceptor.

(ii) Distance between FRET pairs, R: distance between the donorand acceptor fluorophores must fall within the range of ∼1 to10 nm. The FRET efficiency is inversely proportional to the sixthpower of R. Hence, a slight modification in R can significantlyaffect the FRET signals.

iii) Dipole–dipole interaction, �2: donor emission dipole moment,the acceptor absorption dipole moment, and their separationvectors must be in favorable mutual orientation. �2 dependson the angle between the dipole moments of the donor andacceptor in much the same way as the position of a radioantenna can affect its reception. If the donor and acceptor arealigned parallel to each other, the FRET efficiency will be higherthan if they are perpendicular to one another. The degreeof alignment defines the value of �2, which varies between0 (dipole moments are perpendicular to each other) and 4(dipole moments parallel to each other). It is usually assumedto be 2/3, which is the average value integrated over all possibleangles for freely rotating attached-fluorophores.

. Measuring techniques

Numerous methods have been used to measure FRET, suchs, change in donor fluorescence or acceptor emission [37,38].lthough, it is not possible to discuss all of these methods here,

few that are used more often, simple and easy to handle arexplained. Most of the methods mentioned here accommodatehe measurement of both types of FRET processes, i.e. hetero-FRETFRET between two different chromophores) and homo-FRET (FRET

TAC shows the dependency of voltage as a function of time. PMT: photo-multipliertube; CFD: constant fractional discriminator; TAC: time-to-amplitude converter;and MCA: multi-channel analyzer.

between same chromophores). FRET methods can be divided intothe following fundamental categories.

3.1. Donor fluorescence

Monitoring changes in donor fluorescence (either lifetime, i.e.,time-resolved or intensity, i.e., steady-state) in the presence andabsence of acceptor are the most direct method used for the FRETmeasurements. The fluorescence lifetime can be determined witha time-correlated single photon counting (TCSPC) system witha higher accuracy. The advantage of TCSPC system is its higherdetection efficiency, higher signal-to-background ratio, and its sim-plicity. Following schematic diagram (Fig. 3) describes the workingprinciple of TCSPC system.

The most common variants of this approach are FLIM-FRET(a method that measures donor’s fluorescent lifetime, i.e., timespent by the fluorophore at the excited state), based on fluores-cent lifetime imaging (FLIM) [39,40]; and acceptor bleaching (anapproach that measures the intensity of donor fluorescence beforeand after photobleaching acceptors), which is explained later (Sec-tion 3.4) [41]. The fluorescence lifetime is independent of theintensity of the fluorophores, and therefore it is possible to useany cell for FLIM–FRET experiments. Another advantage is thatonly the lifetime of the donor fluorophore has to be measuredand thus, the acceptor fluorophore might have inefficient emis-sion or may even quench the emission. Fig. 4 describes a typicalFLIM setup, where a TCSPC module is coupled to the microscope torecord the fluorescence lifetime and an EMCCD camera for spectralimaging.

Similarly, measuring the fluorescence intensity of a donor beforeand after photobleaching the acceptor is equivalent to measuringthe intensity of the donor in the presence and absence of accep-tors. Bleaching of the acceptors produces an increase in the donor’semission if FRET is occurring. In several cases, it has been foundthat the acceptor quenches the donor fluorescence with a contact-quenching mechanism (which can be corrected on the basis of thevan der Waals radius of the acceptor molecule) [19].

3.2. Acceptor fluorescence

One of the most popular methods used for measuring FRETinvolve 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) 20– 30

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 fluorescence signal for visualization throughed

ifieodtemtasalspTF

3

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Fig. 5. Representation of FRET from fluorescence anisotropy measurements. Donoremission 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: excitationpolarization direction, Black arrow: donor excitation dipole moment, Green arrow:donor emission dipole moment, and Red arrow: acceptor emission dipole moment.During no-FRET, fluorescence from directly excited donor is emitted with a polariza-

yepiece. PD: photo-detector and EMCCD: electron multiplying charge coupledevice.

nvolves acquiring three different images using three fluorescentlter sets. Firstly, a filter set that excites the donor but measuresmission from the acceptor is used to generate a FRET image. Sec-ndly, images obtained with filter sets that measure emission fromonors or acceptors when directly excited are then used to correcthe FRET images. Recently, variants of this method calibrated usingither FLIM–FRET or acceptor-bleaching have been implemented toeasure actual FRET efficiencies [42,43]. The intensity observed in

he corrected FRET image not only encodes information about themount of FRET in the sample, but is also a function of the amount ofample present, the excitation intensity, the excitation wavelength,nd the instrumentation used (filters, objectives, detectors, and theike). An additional advantage of the three-cube approach is itspeed of data acquisition (typically requiring a few seconds as com-ared to minutes for most of the other FRET imaging approaches).his FRET method is applied for time-lapse or three-dimensionalRET imaging in living cells.

.3. Spectral imaging

Recently, a photon-efficient FRET method has been developed,hich monitors changes in the intensity of donors and acceptors

imultaneously using spectral imaging [44]. Information about thearameters like abundance of donors and acceptors, as well asbout the FRET efficiency are encoded in the emission spectra ofonor and acceptor. Earlier, theoretical approaches for measuringRET using spectral imaging have been described and implementedsing two-photon microscopy (a form of microscopy in which twooincident infrared photons, each with only half of the energyequired to excite a particular fluorophore, are used to excite a flu-rophore) [45]. An advantage of using spectral imaging to measureRET is that in addition to yielding FRET efficiency, it also mea-

ures the abundance of donors and acceptors. Because two-photonxcitation is usually implemented with tunable lasers, wavelengthshat efficiently excite both donors and acceptors can be selected.his is important because the judicious selection of excitation

tion similar to excitation causing high polarization. As consequence of FRET, acceptoremission undergoes depolarization compared to the donor excitation because of itsdifferent orientation in the original photoselection plane.

wavelengths for specific samples can maximize the signal-to-noiseratio of both donor and acceptor signals [46]. This FRET methodrequires specialized equipment for spectral imaging, two-photonexcitation, and purified samples of the donor and acceptor fluo-rophores to provide reference spectra.

3.4. Acceptor photobleaching

Acceptor photobleaching method employs selective photo-chemical destruction of the acceptor fluorophore, which, if thetwo fluorophores had previously been physically close enoughto give FRET, results in a release from donor quenching and anincrease in donor emission [47,48]. Technically, the advantage ofthis method is that it reduces the requirements for compensa-tion and calibration associated with standard FRET and can beperformed on normal wide-field microscopes. For accurate FRETmeasurements, this technique requires complete bleaching of theacceptor (which can take several minutes) without bleaching thedonor.

3.5. Fluorescence anisotropy

Fluorescence anisotropy can be used to measure the energytransfer efficiency between the FRET pair directly from the changein anisotropy [7]. When a randomly oriented population of flu-orophores is excited with a linearly polarized light (excitation

polarization), molecules whose absorption/excitation dipoles areoriented parallel to the polarization axis are preferentially excited(Fig. 5A). The resulting anisotropy (r), a measure of the degreeof orientation, is determined by measuring the emission intensity

biology C: Photochemistry Reviews 12 (2011) 20– 30 25

t(

r

atapdalddswoe

r

w(v[

r

S

r

Fm

flssamibitpadbtantti[

tdmbaut

Fig. 6. Schematic diagram of single molecule fluorescence setup. Dotted green andblue lines represent the excitation light with different wavelengths; where as reddotted line is the emission path. As shown in the figure, the emission light is col-

H. Sahoo / Journal of Photochemistry and Photo

hrough vertically (IV) and horizontally (IH) oriented polarizers (Eq.6)).

= IV − IHIV + 2IH

(6)

Depolarization occurs as a result of the emission from thecceptor during FRET, i.e., when the donor transfers its excita-ion energy to a neighboring and proximal acceptor molecule with

different orientation compared to the original photoexcitationlane (Fig. 5B). Particularly for fluorescent proteins, depolarizationue to FRET can be distinguished from that of molecular rotations energy transfer can occur much more rapidly than molecu-ar rotation. By knowing the fluorescence quantum yield of theonor and acceptor (D and A: fluorescence quantum yield foronor and acceptor, respectively) and the anisotropies (rD: emis-ion anisotropy of donor and rA: emission anisotropy of acceptorhen excited through energy transfer), the total anisotropy (rtot)

f the donor–acceptor pair cane be calculated with the followingquation [49],

tot = rDD

tot+ rA

(A

tot

)(7)

here tot is the combined quantum yield of donor and acceptorin case of homo-FRET, it is the same molecule). On substituting thealues of fluorescence quantum yield with rate of energy transfer49], Eq. (7) can be modified as follows;

tot = rD1 + (R0/R)6

1 + 2(R0/R)6+ rA

(R0/R)6

1 + 2(R0/R)6(8)

ubstituting Eqs. (1) and (8) becomes as follows;

tot = rDEFRET

2EFRET − 1+ rA

EFRET − 12EFRET − 1

(9)

In principle, Eqs. (7)–(9) can be used together to estimate theRET efficiency and FRET distance from the fluorescence anisotropyeasurements.Time-resolved fluorescence anisotropy decay and steady-state

uorescence anisotropy are used to measure homo-FRET. Steady-tate intensity or lifetime fluorescence (as mentioned above) is notuitable for homo-FRET measurements as in homo-FRET, intensitynd lifetime values do not change (FRET between two identicalolecules in bidirectional, which leads to no resultant change

n fluorescence intensity or lifetime). In details, energy transferetween identical chromophores located at close enough distances

s possible, provided that there is significant overlap of the absorp-ion and emission spectra, which is the case for most fluorescentroteins including eGFP. The critical Förster distance for eGFPs isbout 4.7 nm [50]. Quantitative analysis of fluorescence anisotropyecays provides information on structural parameters: distanceetween the two interacting chromophores and spatial orienta-ion between the chromophores within dimeric proteins. As fars in vivo system is concerned, fluorescence anisotropy decay isot easy to measure under the microscope and the instrumenta-ions are necessarily sophisticated. Recently it has been shown thatwo-photon excitation steady-state FAIM (fluorescence anisotropymaging microscopy) can be used easily for time-lapse homo-FRET26].

Single molecule fluorescence technique can be coupled withhe above-discussed techniques to measure FRET of individualonor–acceptor pairs. There are several advantages with singleolecule over ensemble measurements. For example, in an ensem-

le measurement the properties of the individual molecules arelways get averaged out; where as single molecule technique buildsp a distribution histogram of individual molecules as a func-ion of time. Also, with its small excitation volume, it reduces the

lected in the same excitation back-focal plane but then using a beam splitter (or,dichroic mirror), it is reflected towards the detectors (APD or EMCCD camera). APDs(avalanche photo diode) are used as detectors to have the fluorescence signal.

background noise mainly due to the Raman and Rayleigh scat-tering, impurities, glass coverslips, optical components, etc. Fig. 6explains the general single molecule setup coupled with a confocalmicroscopy. In this setup, the emission from the sample is collectedwith the same excited plane but then reflected with the beam split-ter (or dichroic mirror) towards the detectors (APD). Before thedetectors, a flip mirror is used to divert the emission path towardsa camera (i.e., EMCCD camera) to collect the individual donor andacceptor images.

4. FRET probes

Optimizing FRET pairs and their attachment to the biomoleculesare the two most-important parameters that can impact the accu-racy of the data interpretation. Fluorophores can be typically andbroadly classified into two different types: intrinsic and extrinsic.Intrinsic fluorophores occur in nature and include aromatic aminoacids (tryptophan, tyrosine, and phenyl alanine), nicotinamide ade-nine dinucleotide (NADH), flavins, and derivatives of pyridoxaland chlorophyll. Most of the intrinsic fluorophores require exci-tation by short wavelength (ultraviolet or blue light), which isoften hazardous to live cells. Additionally, other photophysicalproperties such as, brightness and fluorescence quantum yield aregenerally lower for most of the practical applications of intrinsicfluorophores. These properties made the way for the develop-ment of extrinsic fluorophores, which are typically synthesizedfrom polyaromatic compounds having a conjugated -electron

system. Extrinsic fluorophores are readily attached to the targetbiomolecules in vitro systems, albeit have disadvantages in vivolabeling. However, introduction of labeled molecules into cells hasbeen made possible by utilizing more invasive techniques [51].

26 H. Sahoo / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 12 (2011) 20– 30

Table 1In 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]Tetracysteinea Biarsenic – [67]CLIP-Tag Benzylcytosine – [57]Halo-Tag Aliphatic chloride – [58]SNAP-Tag Benzylguanine – [59]ybbr-Tag Serine sfp-phosphopantetheinyl transferase [68]

Genetical Fluorescent proteinsb GFP – [69]CFP – [70]YFP – [71]RFP – [72]

sed fooresc

4

awlofditadatstLqm

tm

TF

fl

a Tetracysteine tag is mostly used for attaching FlAsH/ReAsH, although it can be ub GFP: Green Fluorescent Protein; CFP: Cyan Fluorescent Protein; YFP: Yellow Flu

.1. Fluorophore labeling methods

Fluorophore labeling is one of the parameters, which candjudge the FRET data accuracy. Reactive fluorescent dyes areidely used to modify amino acids, peptides, proteins (in particu-

ar antibodies), oligonucleotides, nucleic acids, carbohydrates, andther biological molecules. There are different techniques availableor labeling biopolymers. Among the reactive dyes, amine-reactiveyes are most often used to prepare various conjugates for

mmunochemistry, histochemistry, fluorescence in situ hybridiza-ion (FISH), cell tracing, receptor binding, and other biologicalpplications as amino groups are either abundant or easily intro-uced into biomolecules [52–54]. In general, thio-reactive reagentsre frequently used to develop probes for investigating some par-icular protein structure and functions [55,56]. In chemical biology,everal tagged-methods are used to attach the fluorophores to thearget biomolecules, i.e. SNAP, CLIP, Halo (see Table 1) [57–59].astly, the preferred fluorophores should have high fluorescenceuantum yields and retain the biological activities upon the attach-ent to the biomolecules.

There are several methods for fluorophore attachment to the

arget molecules. Below in Table 1 briefly describes commonly usedethods.

able 2orster distances of different FRET pairs (ranging from ultra-violet to visible).

Ultra-violet FRET pairs

FRET pair (D/A) R0/(Å) Ref.

Tryptophan (Trp)/DBO 9 [19]

Tryptophan/Dansyl Chloride 21 [70]Naphthalene/Dansyl Chloride 27 [72]

EDANS/Dabcyl 33 [74]

ATTO 390/ATTO 425 41a

a R0 value obtained from www.atto-tec.com. The above R0 values consider the attacuorophores are not attached to the target molecule.

r other fluorophores as well.ent Protein; and RFP: Red Fluorescent Protein.

Depending upon experimental requirements and existing label-ing techniques (described in Table 1), in vitro labeling can beperformed more readily than that of in vivo. Particularly in thecase of fluorescent protein variants (i.e., Green Fluorescent Protein,GFP), the probes are large enough compared to the movements thatthey report, and quite flexibly attached to the proteins of inter-est allowing for wide differences between the distances of the twoprobes and their attachment points. Additionally, fluorescent pro-tein labeling can induce artifacts via homo-dimerization [73].

Commonly used fluorophores (classified into two differentclasses, e.g., ultra-violet and visible fluorophores) as FRET pairs withtheir characteristic Förster 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 aseither 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 Sulfide). Quantum dotsin particular exhibit critical properties of the fluorophores (suchas high quantum yield and extinction coefficient, brightness, andphotostability). Given the multiplexing capabilities [79–81] and

photostability, QDs exhibit an additional feature of great utility:the number of groups bound to their surface can be varied from1 to few 10 s in a controlled manner. Another striking advan-

Visible FRET pairs

FRET pair (D/A) R0/(Å) Ref.

Alexa 488/Alexa 594 54 [69]Alexa 488/Alexa 647 39 [71]Alexa 555/Alexa 647 51 [73]ATTO 550/ATTO 647 65 [75]Cy3/Cy5 60 [75]GFP3/YFP 56 [76]GFP/RFP 47 [76]CFP/YFP 49 [76]

hment of the FRET pair to the target molecule and thus, it might vary when the

biolog

tsinbammctelasatuDms

5

mactftItoHitfbatpbFb

5

tsbiiclmSugatwFh

H. Sahoo / Journal of Photochemistry and Photo

age is their emission wavelength, which is a function of theirizes [80]. Their use as probes in living cells is rapidly increas-ng due to the advantages and compatibility (and some extent theon-toxicity) with in living systems. As a result of their extremerightness and comparatively long lifetime, QDs are easily detectednd identified visually and by electronic imaging providing singleolecule sensitivity, positional super-resolution [82] and confir-ation of molecular identity (e.g. of substances to which they are

onjugated). As FRET donors, QDs benefit from their large absorp-ion cross-section that increases continuously from their narrowmission bands to the ultra-violet. Thus, despite their relativelyarge size (which can be considered as one of the drawbacks)nd the requirement proximal acceptor(s) to the nanoparticleurface, QDs function efficiently as FRET donors in many bio-nalytical and imaging applications [37,83,84]. And, because ofhe range of the absorbance peak for QDs, which ranges fromltra-violet to violet, these are mostly used as FRET donors.evelopment in QDs with smaller stabilization–conjugation coatsight enable FRET probing at locations removed from the particle

urface.

. Applications

FRET has been utilized as a ‘Spectroscopic Nano-Ruler’ toonitor the structural and conformational perturbations in nano

nd sub-nano scales [35]. In biological science, conformationalhanges are somewhat more demanding in order to understandhe underlying bioprocess mechanisms. In recent times, FRET hasrequently been employed to measure conformations and relatedransitions of the molecule at the molecular level [8,10,11,85].t is noteworthy that the FRET efficiency depends not only onhe distance of the two fluorophores, but also on their relativerientation, i.e., their absorption and emission dipole moments.owever, latter effect is however usually suppressed because

t would be necessary to rigidly attach the donor and accep-or to the molecule of interest. Usually, flexible linkers are usedor fluorophore labeling, thereby averaging over many possi-le orientations, thus limiting the observable distance range to

few nanometers. FRET is an useful biophysical tool to quan-ify molecular dynamics, including protein–protein interactions,rotein–DNA interactions, and protein conformational changesoth in vitro and in vivo systems. Scientific research fields in whichRET has been applied substantially and distinctly are describedelow.

.1. Material chemistry

Fluorescence resonance energy transfer (FRET) has been appliedo cell-material interface for both two and three-dimensional adhe-ion substrates to quantitatively analyze parameters [86]. FRET iseing utilized to quantify several parameters of the cell–material

nterface relevant to cell response, including molecular changesn matrix proteins induced by interactions both with surfaces andells. The mechanism of cell–material interactions at the molecularevel and the quantification of the cell-based nanoscale rearrange-

ent in the material has been investigated by using FRET [87].uch techniques allow both dynamic and 3D analyses that areseful to quantitatively relate downstream cellular responses (e.g.

ene expression) to the composition of this interface. FRET haslso been implemented in core–shell nanoparticles complex sys-ems, which offer different environments for probes associatedith them by different means. Using pyrene and coumarine as a

RET pair, the compartmentalization of the core-shell nanoparticleas been investigated [88].

y C: Photochemistry Reviews 12 (2011) 20– 30 27

5.2. Molecular sensor

FRET has been applied as sensor in various research areas dueto its advantages in investigating the static and dynamic states ofa macromolecule. Recently, different variants of coumarin dye areused as FRET pairs to study the pH regions for sulfadimethoxineand sulfamethizole [89]. Furthermore, the compartmentalized sig-naling of kinase and second-messenger dynamics and the uniquefeatures of genetic abilities with targeting and encoding areunveiled with FRET-based biosensors that allow a real-time track-ing of activity dynamics with high spatiotemporal resolution [90].Where the conventional biochemical processes were unable touncover 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 [19,35,93–95], carbo-hydrates [96], and biomolecules [97] are used as spacer betweenthe donor and acceptor to reveal there rigidity depending on chainlength and also different fluorophores are tried with the same set ofspacers to have more accuracy with the FRET distances [94,74]. Fol-lowing figure (Fig. 7B) displays the FRET dependency on the lengthof a peptide chains. In this case, glycine-serine (Gly-Ser) units areused as the separation between the FRET pair (Trp and DBO) asshown in Fig. 7A [19].

Study of biopolymers of different chain lengths with amino acidsas the spacer provides an idea about the flexibility at differentregions in a three-dimensional protein structure. To understandthe detailed role of carbohydrates on bioprocesses, FRET has beenused to investigate the conformational changes in carbohydratesby labeling the fluorophore through biotin-streptavidin chemistry[96].

5.4. Biomolecular interactions

FRET and FLIM have been applied extensively to biomolecularinteraction studies: in the analysis of protein–protein interac-tions with high spatial and temporal specificity (e.g. clustering),in the study of conformational changes, in the analysis of bindingsequences, and in applications such as high-throughput screen-ing [98–100]. Recent advances in microscopy have allowed thedevelopment of new methods to analyze protein–protein interac-tions at very high resolution in both fixed and live cells [101–103].Additionally, molecular interactions and conformational changesof various proteins involved in the regulation of cell adhesionand motility have been investigated [104]. FRET measurement ofSecretin docking with its receptor provides strong evidence for theorientation 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 been employed to determine the biomolecular dynam-ics and conformations occurring in biological pathways. Forexample, RNA folding [106], small DNA bending angles, the func-tional meaning of which may be just as important as of largebends (DNA bending) and DNA–protein interactions [107]. Fig. 8displays a general scheme that shows the extraction of protein fold-ing intermediates and their dynamic information from the distance

distributions through FRET analysis. Basically, based on the FRETdistances between different sites (of interest) in a protein, charac-terization of different intermediate states can be carried out. Thedistance distribution probabilities in different intermediates reveal

28 H. Sahoo / Journal of Photochemistry and Photobiolog

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 (wheren = 0, 1, 2, 6, and 10). Where, Trp (at N-termini) and DBO (at C-termini) serve as donorand acceptor, respectively (taken from J. Phys. Chem. B 111 (2007) 2639–2646). (B)Decrease in donor fluorescence intensities, which alternatively indicate the FRETeTT

tt

m

FmmftT

fficiency, as a function of number of intervening amino acids (n). Reference peptide:rp-(Gly-Ser)6–COOH.he spectra are adapted from Ref. [19].

he compactness of the protein and consequently the conforma-ional changes during the folding process.

Recently, single molecule FRET (sm-FRET) of different confor-ations of the tumor suppressor gene (p53) study shows that the

ig. 8. Protein folding funnel. FRET analysis showing different energy-related inter-ediates during protein folding pathway. The unfolded states are comparativelyore flexible than the native 3-dimensional tertiary state. Unfolded states with dif-

erent energies folds to a rigid and native state through several intermediate stateshat differs in their energy distributions.he folding funnel is taken from Ref. [9].

y C: Photochemistry Reviews 12 (2011) 20– 30

N-terminal domain weakly binds to the DNA binding domain of thep53. Together with time-resolved FRET (tr-FRET), different confor-mations of the different domains in p53 were monitored, which aremostly overlooked in ensemble FRET technique [13]. FRET analy-ses revealed that the interaction between Fibronectin domains (IIIA and III B) exposes the binding sites in solution through conforma-tional changes in Fibronectin III [108]. Besides the conformationalstudies, 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 fluorescence, which was laterimplemented in protein folding by Hochstrasser and coworkers[110] and Weiss and coworkers [8]. A unique aspect of the freediffusion experiments on single molecules is that structural anddynamic properties of the subpopulation of unfolded molecules canbe separately investigated at equilibrium in the presence of a largeexcess of folded molecules, where the ensemble averaged proper-ties are dominated by the folded state. A consistent finding in all ofthe free diffusion experiments is that the overall size of the unfoldedprotein, as obtained from the FRET efficiency-determined, increasescontinuously with increasing in denaturant concentration. Thisbehavior, first 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 domainof protein A [41], the immunity protein Im9 [15] and the prion-determining domain of Sup35 [114].

5.6. Host–pathology interactions

Given the advantages and compatibility of FRET with other tech-niques, its use in understanding host–pathogen interplay has, todate, been surprisingly limited [115]. FLIM–FRET facilitates theuse of FRET to measure the physical distance between donorand acceptor fluorophores, a technique utilized by Latz et al. todecipher signaling associated with immune recognition of CpGDNA, a pathogen-associated molecular pattern (PAMP), by the toll-like receptor, TLR9 [116]. Fluorescence lifetime imaging is rapidlybecoming 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 alsoallow the subcellular localization processes like, binding of SH2(Src Homology 2) domain of Hck, a tyrosine-protein kinase, totyrosine-phosphorylated cytoplasmic domain of CEACAM3 (carci-noembryonic antigen-related cell adhesion molecule 3) in intactcells, during bacterial infection [118]. FRET-based assays are valu-able tools to resolve bacteria-induced protein–protein interactionsin the context of the intact host cell.

5.7. Drug and ligand screening

TR-FRET (time-resolved FRET) brings together the low back-ground benefits of TRF with the homogeneous assay format ofFRET. This powerful combination provides significant benefitsto drug discovery researchers including assay flexibility, relia-bility, increased assay sensitivity, higher throughput and fewerfalse positive/false negative results. Compared to other bindingassays, the polarization [119] and HTRF (high-throughput TR-FRET)[120] binding assays are nonradiaoactive, therefore safer toperform, yet very sensitive and homogeneous, therefore easierand faster to automate. These methods are thus suitable for effi-cient drug high-throughput screening procedures and can easily beapplied to other G protein-coupled receptor models. FRET-basedassay are used to screen a G-protein-coupled receptor-focused

library of fluorescent compounds on the human eGFP-taggedapelin receptor [121]. A series of fluorescent ligands designed forvasopressin and oxytocin G protein-coupled receptors was syn-thesized and characterized to develop fluorescence polarization or

biolog

h[

5

d(abrdalocosbaqcfFtp(

6

Fdvt[olmIomsm

teispBa[oaatattimaep

H. Sahoo / Journal of Photochemistry and Photo

omogeneous time-resolved fluorescence (HTRF) binding assays122].

.8. Lipid membrane

Loura and co-workers have reviewed the application FRET inetecting and characterizing the phase separation in lipid bilayersboth in model systems and in cell membranes) [123]. This review islso focused on models describing the rate and efficiency of FRET foroth uniform probe distribution and phase separation, and recentlyeported methods for detection of membrane heterogeneity andetermination of phase boundaries, probe partition coefficientsnd domain size. FRET is also applied to characterize single-phaseipid systems, gel/fluid phase separation, liquid ordered/liquid dis-rdered phase separation (lipid rafts), complex systems containingeramide and cell membranes are presented to illustrate the wealthf information that can be inferred from carefully designed FRETtudies of membrane domains. The intermembrane contact areasetween single unilamellar vesicles and planar supported bilayersre quantified using FRET as basic tool [124]. In a recent review,uantification of interaction of membrane proteins with biologi-ally relevant lipid molecules using FRET methodologies has beenocused in details [125]. In the field of protein–lipid interactions,RET has also been extensively used to characterize and quan-ify partition of membrane proteins to lipid membranes [126] andarticular lipid phases [127], and most notably to liquid-orderedraft-like) phases [128].

. Conclusions and outlook

With the recent advancements in optical instrumentations,RET along with other existing optical techniques is used to revealynamics and interactions of fluorescently labeled molecules withery high precision. Thus, FRET is becoming considerably impor-ant in combination with single molecule fluorescence microscopy8]. Because of the non-invasiveness, simple, robust, and designf genetically encoded fluorophores, FRET is of tremendous use inife science research. FRET also enables the quantitative measure-

ent of molecular interactions in living cells and even in organisms.n this review, the pitfall of FRET data interpretation in termsf the fluorophore selection and their attachment to the targetolecules has been discussed. Also the use of different excitation

chemes, which can bring the accuracy with regards to cross-talk, isentioned.Major new FRET developments in the area of fluorescence life-

ime determinations, either in the time or frequency domain willxploit the techniques like, FLIM (or FLI, fluorescence lifetimemaging), which can be combined with multispectral, polarization-ensitive, and optical-sectioning modalities and as such offers therospect of a ‘do-it-all’ form of fluorescence microscopy [129].esides, combination of FRET with techniques like single-moleculend FCS exposes the dynamic processes in biomolecular processes130]. Also, the emergence of numerous new techniques basedn parameter modulation and perturbation, permitting the reli-ble phase-sensitive detection of extremely low FRET signals arenticipated in the years to come. Significant driving technology inhe areas of illumination sources (LEDs, Laser Emitting Diodes),dvanced optical imaging techniques, and improvements in theheoretical framework dictating modes of data (image) acquisi-ion and analysis will make FRET a more powerful technique

n investigating the biological fundamental processes. Further-

ore, efforts devoted to facile introduction of diverse organicnd nanoparticle probes into cells is expected to exploit andstablish mixed kind of FRET pair (organic and nanoparticlerobes).

y C: Photochemistry Reviews 12 (2011) 20– 30 29

Acknowledgments

Dr. Jan K. Rainey (Dalhousie University, Canada), Dr. DaniloRoccatano (Jacobs University Bremen, Germany), and Dr. FangHuang (China University of Petroleum-Shandong, China) are highlyacknowledged 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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