Optimization of Pairings and Detection Conditionsfor Measurement of FRET between Cyan andYellow Fluorescent Proteins

Mark A. Rizzo,1 Gerald Springer,2 Katsuhisa Segawa,3 Warren R. Zipfel,4 and David W. Piston2,*

1University of Maryland School of Medicine, Baltimore, MD 21201, USA2Department of Molecular Physiology and Biophysics, 735 Light Hall, Vanderbilt University, Nashville, TN 37232, USA33-204 Murayamadainijutaku, 1-2-46 Fujimicho Higashimurayama-shi, Tokyo 189-0024, Japan4Applied & Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, NY 14853, USA

Abstract: Detection of Förster resonance energy transfer ~FRET! between cyan and yellow fluorescent proteinsis a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellowfluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRETbetween monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greaterFörster distance. We also compared several live cell microscopy methods for measuring FRET. The greatestcontrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectralimaging. However, this method is not appropriate for establishing the presence of FRET without extra controls.Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurementsare difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuringFRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomesprone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was alsodetected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method isexceptional for screening purposes, because it provides high contrast for discriminating FRET.

Key words: FRET, GFP, two-photon microscopy, confocal microscopy, fluorescence spectral imaging, anisot-ropy, lifetime imaging

INTRODUCTION

Förster resonance energy transfer ~FRET! between two flu-orophores is observed over distances less than 10 nm ~Förster,1948!. Because FRET efficiency is steeply dependent on thedistance separating the FRET pair and the relative orienta-tion of the fluorophores, FRET can to be used to detectchanges in protein conformations or protein–protein inter-actions. However, the sensitive nature of FRET also presentstechnical challenges to its measurement, especially whenapplied to complicated experimental systems that are notwell suited for detecting FRET. This is particularly true formeasurement of FRET between two fluorescent proteins~FPs! using fluorescent microscopes.

FP-FRET has been widely incorporated into two gen-eral types of living-cell-based assays, with each applicationpossessing unique challenges. The first strategy incorporates

measurement of intermolecular FRET between FPs conju-gated to opposing ends of a protein or an environmentallysensitive protein domain ~Miyawaki et al., 1997; Pearceet al., 2000; Honda et al., 2001; Ting et al., 2001; Zhanget al., 2001!. Although this general strategy has seen widesuccess, utilization of FPs in these assays provides specialchallenges related to the properties of FPs themselves. Thelarge size of FPs ~4.2 nm long by 2.4 nm diameter barrel;Ormo et al., 1996! occupies much of the useful FRETdistance ~Patterson et al., 2000!, resulting in FRET efficien-cies less than 30%. In addition, FPs come in a wide range ofbrightness. For example, the most popular donor FP, ECFP,is fivefold less bright than the most popular FRET acceptor,EYFP. Finally, the spectra of FPs are quite broad and greatlyoverlapping, which hinders both exclusive donor excitationand separation of donor and acceptor emissions. All ofthese factors severely limit the amount of contrast that canbe obtained using an intramolecular FRET based strategy,with even the best biosensors reporting a small change inFRET that barely exceeds the noise of many imaging sys-tems ~Swedlow et al., 2002!. These complications are alsorelevant to the use of intermolecular FRET to measureprotein–protein interactions between two FP-labeled pro-teins. In addition, the oligomeric nature of FPs provides

Note: M. Rizzo and K. Segawa performed this research at VanderbiltUniversity ~same address as Piston!. W. Zipfel performed research atCornell University.Received September 1, 2005; accepted October 26, 2005.*Corresponding author. E-mail: dave.piston@vanderbilt.edu

Microsc. Microanal. 12, 238–254, 2006DOI: 10.1017/S1431927606060235 Microscopy AND

Microanalysis© MICROSCOPY SOCIETY OF AMERICA 2006

additional complications when they are used in intermolec-ular FRET studies and makes these experiments more diffi-cult to interpret. Even the most commonly used FPs, ECFPand EYFP, are weakly dimeric ~Zacharias et al., 2002!, whereasnon-Aequorea derived FPs, such as dsRed ~Wall et al., 2000!and HcRed ~Gurskaya et al., 2001!, tend to be obligateoligomers. Thus, FRET experiments that incorporate FPsare difficult to design and execute.

Several strategies have been proposed for overcomingthe difficulties associated with FP-FRET experiments. Theseinclude the application of algorithms that correct for emis-sion crosstalk ~Youvan et al., 1997; Gordon et al., 1998; Xia& Liu, 2001; Hoppe et al., 2002; Zal & Gascoigne, 2004!,design and discovery of FPs with improved properties ~Heim& Tsien, 1996; Karasawa et al., 2004; Rizzo et al., 2004;Shaner et al., 2004; Nguyen & Daugherty, 2005!, and devel-opment of improved microscopy techniques ~Erickson et al.,2001; Harpur et al., 2001; Zimmermann et al., 2002; Mat-theyses et al., 2004; Rizzo & Piston, 2005!. Because thesenumerous and diverse methods for imaging FP-FRET can-not be simultaneously applied, a useful comparison of avail-able FP-FRET partners and methods can provide a basis forselecting the most suitable conditions for a particular FRETexperiment. Therefore, we have created a set of conjoinedFRET pairs and compared their FRET efficiency. We alsocalculated the Förster distances and determined optimizedsingle-photon and two-photon excitation parameters foreach pairing. The most efficient conjoined pair used in thisstudy, mCerulean to mVenus, was then expressed in livingcells to compare several strategies for FRET detection. Thehighest contrast for intramolecular FRET imaging wasobtained by utilizing optimized donor excitation in combi-nation with ratiometric spectral imaging. Considering tech-niques more suitable for intermolecular FRET experiments,fluorescence lifetime imaging microscopy ~FLIM! reportedthe most accurate FRET efficiencies. However, donor de-quenching experiments consistently overestimated the FRETefficiency as a result of acceptor absorption of the initialexcitation energy. Finally, detection of sensitized emissionanisotropy was found to be the highest contrast techniquefor distinguishing the presence of FRET.

MATERIALS AND METHODS

Recombinant DNA Procedures

The monomeric mutation, A206K ~Zacharias et al., 2002!,was introduced into Aequorea-derived FPs using theQuikchange site-directed mutagenesis kit ~Stratagene, LaJolla, CA! with the following primer sets: mEGFP ~sense5'-CTGAGCACCCAGTCCAAACTGAGCAAAGACCCC-3',antisense 5'-GGGGTCTTTGCTCAGTTTGGACTGGGTGCTCAG-3'!, mVenus ~sense 5'-CTGAGCTACCAGTCCAAACTGAGCAAAGACCCC-3', antisense 5'-GGGGTCTTTGCTCAGTTTGGACTGGTAGCTCAG-3'!, mCerulean ~sense 5'-

GCACCCAGTCCAAGCTGAGCAAAGA-3 ', antisense 5 '-TCTTTGCTCAGCTTGGACTGGGTGC-3'!, and mCitrene~sense 5 '-CTGAGCTACCAGTCCAAACTGAGCAAAGACCCC-3', antisense 5'-GGGGTCTTTGCTCAGTTTGGACTGGTAGCTCAG-3'!. FPs not already in the C1 vector wereamplified by PCR and subcloned into the C1 vector follow-ing excision of EGFP from the pEGFP-C1 vector. mVenuswas amplified using sense primer 5'-TTTACCGGTCGCCACCATGGTGAG-3' and antisense primer 5'-TTCTTACTTGTACAGCTCGTCCATGCCG-3' and subcloned into the C1vector using AgeI and BsrGI restriction sites. CopGFP wasamplified from the pCop-Green-C plasmid ~Evrogen, Mos-cow, Russia! ~sense primer 5'-TTTAGTGAACCGTCAGATCCGC-3 ', antisense primer 5-AATCCGGAGGCGAAGGCG-3'! and subcloned into the C1 vector using NheI andBspEI restriction sites. Generation of phiYFP-C1 andphiYFPm-C1 were preceded by the introduction of silentmutations to remove key restriction sites in the cDNA. TwoBsrGI sites ~1029Gr C: sense primer 5'-TGGCAGCGTCTACAATCGCG-3', antisense primer 5'-ACGCGATTGTAGACGCTGCC-3' ; 1122G r C sense primer 5'-CCCACTGCCTCTACATCTGG-3', antisense primer 5'-CCAGATGTAGAGGCAGTGGG-3'! and a BglII site ~1173C r T senseprimer 5 '-CCTTCAAGATTTGCCACGAG-3 ', antisenseprimer 5'-CTCGTGGCAAATCTTGAAGG-3'! were first re-moved using the Quikchange site-directed mutagenesis kit.To generate phiYFPm, the same approach was used tointroduce three additional mutations into phiYFP ~K221T:sense primer 5 '-TATGAGCCTGACGGAGACCGTGC-3 ',antisense primer 5'-GCACGGTCTCCGTCAGGCTCATA-3' ; T170A: sense primer 5'-CCACGAGATCGCCGGCAGCAA-3', antisense primer 5'-TTGCTGCCGGCGATCTCGTGG-3' ; N21D: sense primer 5'-GATGGAGGGCGATGTGGATGG-3', antisense primer 5'-CCATCCACATCGCCCTCCATC-3'!. The modified phiYFP and phiYFPm were thensubcloned into the C1 vector ~sense primer: 5'-AAAGCTAGCGCTACCGGTCGCCACC-3', antisense primer 5'-TTTTCCGGACAGGTAGGTCTTGC-3' ; NheI–BspEI fragment!.

A series of N3 vectors were generated from thepEGFP-N3 by replacing EGFP with PCR products amplifiedfrom C1 vectors using BamHI and BsrGI restriction sites.For Aequorea-derived FPs, sense primer 5'-AAGGATCCCCACCGGTCGCC-3' and antisense primer 5'-TTGAGCTCGAGATCTGAGTCCGG-3' were used. PhiYFP and phiYFPmwere amplified using sense primer 5'-AAGGATCCCCACCGGTCGCC-3' and antisense primer 5'-AAGAACATGTACAGGTAGGTCTTGC-3'. CopGFP was amplified using senseprimer 5'-AAGGATCCCCACCGGTCGCC-3' and antisenseprimer 5'-TTCTTGTACATGGCGAAGGCGATG-3'.

His-tagged monomeric FPs were generated by sub-cloing from the C1 vectors into the pQE9-N1 vector ~Rizzoet al., 2004! using NheI and HindIII restriction sites. His-tagged conjoined FRET pairings were generated by firstsubcloning donor fluorophores ~mCer, mEGFP, and copG!from the N3 vector into the bacterial expression plasmidpQE-N1-NotI ~Rizzo et al., 2004! using NheI and NotI

Comparison of GFP FRET Pairs 239

restriction sites. Acceptor fluorophores were added either byligating the NheI–BglII fragment from C1 vectors to aNheI–BamHI digested plasmid to generate a short linkerwith the sequence SGLRSPPVAT or by ligating a NheI–BamHI fragment from a C1 vector to the NheI–BglII di-gested plasmid to generate a long linker with the sequenceSGLRSRAQASNSAVDGTAGPGSRAQASNSAVDGTAGPGSPPVAT. Mammalian expression coupled FRET pairs weregenerated by ligating the NheI–BglII fragment from C1vectors to a NheI–BamHI digested N3 donor plasmid togenerate a short linker or by ligating a NheI–BamHI frag-ment from a C1 vector to NheI–BglII digested N3 donorplasmid to generate the long linker.

Restriction enzymes were obtained from New EnglandBiolabs ~Beverly, MA! and ligations were performed usingQuickLigase ~New England Biolabs!. DH5a bacteria wereused to maintain mammalian expression constructs, andXL1-Blue bacteria were used to maintain plasmids contain-ing bacterial expression promoters. DNA isolation was per-formed using QIAprep Spin Miniprep Kit ~Qiagen Inc.,Valencia, CA! for plasmid purification and QIAquick GelExtraction Kit ~Qiagen Inc.! for purification from agarosegels. Sequences were verified by restriction digest and se-quencing reactions performed by the Vanderbilt-IngramCancer Center DNA Sequencing Shared Resource.

Protein Purification and Spectroscopy

His-tagged recombinant proteins were prepared usingM15~pRep4! cells ~Qiagen Inc.! and purified using Ni-NTAagarose resin as previously described ~Rizzo et al., 2004!.Protein concentrations were quantified using the AdvancedProtein Reagent ~Cytoskeleton Inc., Denver, CO!, and SDS-PAGE. Sample purity was assessed by SDS-PAGE analysisand determined to be greater than 95%.

Steady-state fluorescence measurements were acquiredusing a T-format spectrofluorometer ~Photon TechnologyInternational, Inc., Lawrenceville, NJ!. Absorbance measure-ments were acquired using the 8453 UV-Visible system~Hewlett-Packard, Palo Alto, CA!. Extinction coefficientswere calculated by applying Beer’s law to the peak absor-bance ~in TE buffer @10 mM Tris-HCl, 1 mM EDTA, pH 8.0#!of three separate concentrations of protein taken in quadru-plicate. Linear regression analysis was used to generateextinction coefficients from the slope of protein concentra-tion versus absorbance plots. Molar concentrations werecalculated from the theoretical protein sequence using thePeptide Properties calculator ~http://www.basic.nwu.edu/biotools/ProteinCalc.html!. Quantum yields were deter-mined from integrated fluorescence spectra taken from anequal absorbance of a fluorescein reference standard ~quan-tum yield � 0.95! ~Molecular Probes, Inc., Eugene, OR! inTE buffer ~pH 8.0!. Measurements were performed in qua-druplicate on solutions with equivalent absorbance ~,0.05!and corrected for background fluorescence and differencesin excitation intensity. For two-photon anisotropy measure-

ments, excitation light from a Mira 900 Ti:Sa laser ~Coher-ent Inc., Santa Clara, CA! was directed into the fluorometerand focused into the cuvette using a 60-mm lens. A half-wave plate was used to modify the polarization of theexcitation beam when required, and fluorescence emissionwas passed through 2-mm BGG22 glass ~Schott, Elmsford,NY! prior to collection.

Fluorescence lifetimes were acquired in TE buffer~pH 8.0! using a frequency doubled Ti:Sa laser ~CoherentInc.! and a time-correlated single photon counting module~Edinburgh Instruments LTD., Livingston, UK!. Fluores-cence lifetime measurements were collected at 475 nm ~2-mmslits, ;8-nm bandwidth! using 425 nm excitation formCerulean in addition to ECFP:EYFP, mCerulean:mVenus,mCerulean:mCitrene, and mCerulean:phiYFPm conjoinedpairs. A collection wavelength of 470 nm was used formCerulean:mEGFP pairs ~425 nm excitation!, and 465 nmlight was collected for mCerulean:copGFP pairs. A wave-length of 445 nm was used for excitation of EGFP, mEGFP,and copGFP in addition to mEGFP:mVenus, copGFP:mVenus, mEGFP:mCitrene, and copGFP:mCitrene con-joined pairs ~495 nm emission!. For mCitrene and mVenus,445 nm excitation was used with 530 nm collection. Col-lection at 535 nm was used for phiYFP and phiYFPmdetection ~445 nm excitation!. Magic angle conditions ~08polarized excitation, 54.78 polarized collection! were used toeliminate the effect of polarization. Curve fitting was per-formed using the F900 software ~Edinburgh InstrumentsLTD!. Reported values are the average of four measurements.

Cell Culture

COS-7 cells were cultured in Dulbecco’s Modified Eagle’s Me-dium ~Invitrogen Corporation, Carlsbad, CA! supplementedwith 10% fetal bovine serum and antibiotics as previouslydescribed ~Rizzo et al., 2004!. Plasmid DNA was deliveredinto COS7 cells either by electroporation or transfection.Square wave electroporation ~10 � 50-ms pulses, 300 V,500-ms intervals, 4-mm gapped cuvette! was performed asdescribed ~Rizzo et al., 2004! prior to seeding in 35-mm dishescontaining No. 1 glass coverslips ~MatTek Corporation, Ash-land, MA! for microscopic observation. For coexpression ofunlinked mVenus and mCerulean, 9-mg mVenus-C1 and3-mg mCerulean-N3 were cotransfected into COS-7 cellsusing Fugene transfection reagent ~Roche Diagnostics Corpo-ration, Indianapolis, IN! according to the manufacturer’sprotocol.

Fluorescence Microscopy

Spectral imaging and linear unmixing was performed aspreviously described ~Rizzo et al., 2004! using an LSM510META system ~Carl Zeiss MicroImaging, Inc., Thornwood,NY!. Imaging of COS-7 cells was performed with 458-nmexcitation provided by an Ar laser and a 63�, 1.4 NAapochromat objective lens. Multiphoton excitation was pro-vided by a Mira 900 Ti:Sa laser ~Coherent Inc.!.

240 M.A. Rizzo et al.

FLIM was performed using a modified BX61WI up-right Fluoview 300 laser scanning microscope ~OlympusAmerica Inc., Melville, NY! in conjunction with a femto-second pulsed Mira 900 Ti:Sa laser ~Coherent! and fluores-cence lifetime imaging hardware and software from Beckerand Hickl ~Berlin, Germany!. Attenuation of the laser emis-sion ~'76-MHz repetition rate! was done with a compen-sated attenuator ~925B; Newport, Irvine, CA!. A periscopewas used to direct the excitation beam into the scan controlunit of the microscope. This beam was then directed to a20� water immersion lens ~XLUMPlanFl 0.95 NA; Olym-pus America Inc.!, where it was focused to a diffractionlimited spot. Fluorescence emission was reflected backthrough the same objective and collected in a non-descannedconfiguration after reflection from a dichroic mirror~675dcxru, Chroma Technology, Rockingham, VT!mountedin a reconfigured arrangement in the microscope’s filterturret. After passing sequentially through an IR blockingfilter ~BGG22; Schott!, a narrow band filter ~D480/40M;Chroma Technology, Rockingham, VT! and a GRADIUMplanoconvex focusing optic ~GPX085LR14; Newport! singlefluorescent photons were detected by an externally mountedmicrochannel plate photomultiplier tube ~MCP-PMT!~R3809U-50; Hamamatsu, Bridgewater, NJ!. The MCP-PMTwas coupled to the microscope with a shutter ~Uniblitz;Rochester, NY!. The high-voltage power supply ~FuG-HCN-14-3500-A-US; Becker and Hickl!, the output amplifier~HFAC-26, 26-dB, 1.6-GHz preamplifier; Becker and Hickl!,and the overload protection of the MCP-PMT are all con-trolled by Becker and Hickl’s instrument control softwareand associated electronic modules. Signals from the MCP-PMT were routed to the start channel of a SPC-830 time-correlated single-photon counting module ~TCSPC! ~Beckerand Hickl!, which operates in reverse start–stop mode. Trig-gering in this mode was done using the laser photodiodeoutput signal from the control unit of the Mira 900. TheSPC-830 module was regulated using measurement and con-trol software ~SPC-M! from Becker and Hickl. The line andframe clocks from the scan controls of the Olympus BX61WIwere routed to the SPC-830. The pixel clock generated bythe Fluoview’s scan controls was not used during lifetimeimaging. A pixel clock on the SPC-830 that was controlledby the SPC-M was used instead. Data analysis of the lifetimeimages was done using SPCImage ~Becker and Hickl!.

Fluorescence polarization microscopy was performedusing an LSM410 ~Carl Zeiss MicroImaging! in conjunctionwith a femtosecond pulsed Mira 900 Ti:Sa laser ~Coherent!.A half-wave plate ~Newport! oriented at 458 was used toadjust polarization of the beam when required. Fluores-cence emission was passed through 3-mm BGG22 coloredglass, a 515–540-nm bandpass filter, and a polarized filterprior to collection.

Calculations

FRET efficiency ~E ! was calculated from the fluorescencelifetime measurements using the equation

E � 1 �tDA

tD

, ~1!

where tD is the fluorescence lifetime of the donor and tDA isthe fluorescence lifetime of the donor in the presence of theacceptor ~Clegg, 1992!.

Optimized wavelengths for single photon excitationwere calculated by plotting

FD~lex !

� FAs~l!

FDs~l!

�FAL~l!

FDL~l!�

as a function of excitation wavelength, where FD is thefluorescence of the donor alone, FAS

is the acceptor fluores-cence of the short linker conjoined FRET pair, FDS

is thedonor fluorescence of the short linker pair, FAL

is theacceptor fluorescence of the long linker pair, and FDL

isthe donor fluorescence of the long linker donor pair. Fortwo-photon measurements, the optimal wavelength was es-timated from the peak value of plotting ~FD

2p !2/FA2p versus

the excitation wavelength, where FD2p is the two-photon

cross section of the donor, and FA2p is the two-photon cross

section of the acceptor.Optimized wavelengths were calculated from data ob-

tained using the following conditions on a T-format fluo-rometer: 475 nm donor emission and 525 nm acceptoremission for ECFP:EYFP, mCerulean:mCitrene, andmCerulean:mVenus pairs; 475 nm donor emission and505 nm acceptor emission for mCerulean:mEGFP pairs;470 nm donor emission and 500 nm acceptor emission formCerulean:copGFP pairs; 475 nm donor emission and535 nm acceptor emission for mCerulean:phiYFPm pairs;and 495 nm donor emission and 530 nm acceptor emissionfor mCitrene:mEGFP, mEGFP:mVenus, copGFP:mCitrene,and copGFP:mVenus pairs. Polarizers were set at magicangle conditions to eliminate the effects of polarization.

Förster distances, r0, were calculated by the method ofPatterson et al. ~2000! from the Förster equation

r06 �

9

4~2p!52303

Nk2fD n�4�

0

`

FD~l!«A~l!l4dl, ~2!

where N is Avogadro’s number, k2 is orientation factor, fD

is the quantum yield of the donor, and n is the refractiveindex. The constants preceding k2 have a value of 8.786 �10�11 mol L�1 cm nm2. Within the spectral overlap integral,FD~l! is the fluorescence emission intensity normalizedsuch that its integral over wavelength is unity, and «A is theextinction coefficient of the acceptor. Förster distances werecalculated assuming randomization of dipole orientations~k2 equal to 2/3!, and n was set at the value used for waterat 258C ~n � 1.3342!.

Comparison of GFP FRET Pairs 241

The overlap integral was calculated from normalizeddonor emission and acceptor excitation spectra. Donoremission spectra were obtained using the following con-ditions: 425 nm excitation, 440–600 nm emission formCerulean and ECFP; 440 nm excitation, 455–600 nmemission for copGFP; 450 nm excitation, 465–600 nm emis-sion for mEGFP; 450 nm excitation, 465–620 nm emissionfor mCitrene, mVenus, and EYFP; 455 nm excitation, 470–620 nm emission for phiYFPm. Acceptor excitation spectrawere obtained using the following conditions: 350–485 nmexcitation, 500 nm emission for mCerulean and ECFP;350–525 nm excitation, 545 nm emission for mEGFP andcopGFP; 350–545 nm excitation, 565 nm emission for EYFP,mCitrene, and mVenus; and 350–560 nm excitation, 575 nmemission for phiYFPm.

Two-photon cross sections were obtained as previouslydescribed ~Zipfel et al., 2003! and calculated as follows. Thenumber of fluorescence photons emitted per second from afocused mode-locked laser is

F � s2 Cj�g^I0 &2�

�`

`

psf 2 dV�, ~3!

where s2 is the two-photon action cross section ~product ofthe quantum yield and the absolute cross section!, C theconcentration, j the integrated ~over wavelength! systemcollection efficiency of the particular emission lineshape, gthe second-order coherence ~which has the laser repetitionrate and pulsewidth dependence in it!, ^I0&

2 the squaredpeak intensity, and the last term is the normalized squaredillumination point spread function integrated over all space.The term ^I0 &

2*�`` psf 2 dV is the average intensity which is

linearly proportional to the measure power. If the fluores-cence is plotted versus average power for values belowsaturation, the slope ~S � s2jgC! can be obtained. Thecross section is calculated by ratioing the slope of theunknown I2 curve with the I2 slope of a compound with aknown cross section:

Sunk

Sref

�s2, unkjunk Cunk

s2, ref jref Cref

~4!

� s2, unk � � Sunk

Sref�� jref

junk�� Cref

Cunk�s2, ref .

The integrated collection efficiency j is calculated numeri-cally as

j ��Q~l!T ~l!F~l! dl, ~5!

where Q is the PMT quantum efficiency, T the filter anddichroic transmission lineshape, and F the normalized sam-ple fluorescence lineshape.

Fluorescence anisotropy was calculated from data col-lected using the two-channel method, using vertically polar-ized excitation light and simultaneous collection of parallel~I5! and perpendicularly polarized emission ~I4!. Fluores-cence anisotropy ~r! was then calculated as

r �I5� I4

I5� 2I4. ~6!

To correct for polarization bias in the instrument, thehorizontally polarized excitation was used for calculation ofa correction factor ~Lakowicz, 1999!.

Fluorescence Polarization Imaging

To calculate fluorescence anisotropies using a fluorescencemicroscope, several corrections must be applied to accountseparately for the instrumentation bias of the instrumentand for the effects of a high NA objective lens on observa-tion of fluorescence polarization as defined by Axelrod~1979!. A total of four images were taken as described~Blackman et al., 1996; Rocheleau et al., 2003; Rizzo &Piston, 2005!: Vertically polarized excitation of the donorwas used in combination with capture of both horizontal~VH ! and vertical ~VV ! polarizations, and horizontallypolarized excitation was used in combination with captureof both horizontal ~HH ! and vertical ~HV ! polarizations. Acorrection factor ~g! for instrumentation polarization biaswas then calculated by solving the equation

VV

g � VH�

g � HH

HV. ~7!

The polarization of emitted light at the focal spot can bedescribed along three axes, Ix , Iy, and Iz, and becomes mixedsuch that the images collected at parallel ~VV ! and perpen-dicular ~VH ! geometries can be expressed essentially asdescribed by Axelrod ~1979!:

VV � Kc Ix � Kb Iy � Ka Iz

VH � Kb Ix � Kc Iy � Ka Iz , ~8!

where

Ka � 31�~2 � 3 coss0 � cos3 s0 !

Kb � 112_~1 � 3 coss0 � 3 cos2 s0 � cos3 s0 !

Kc � 41�~5 � 3 coss0 � cos2 s0 � cos3 s0 ! ~9!

and angle s0 is calculated from the NA of the objective lensfrom the equation NA � n sins0. An objective lens with anNA of 1.3 was used for collection and a value of 1.37 was

242 M.A. Rizzo et al.

used for the refractive index ~n! of the cytoplasm ~Brun-sting & Mullaney, 1974!. If the fluorophore is randomlyoriented, Iy � Iz, thus allowing solving for vectors Ix and Iy.The anisotropy, r, can then be calculated as follows:

r �Ix � gIy

Ix � 2gIy

. ~10!

Images were processed using ImageJ software ~http://rsb.info.nih.gov/ij/index.html!.

RESULTS

Comparison of FP FRET Pairs

ECFP and EYFP is the most widely used pair for FP-FRET–based assays, but the development of new techniques andreagents has led to use of optimized variants or even uncon-ventional pairings ~Harpur et al., 2001; Clayton et al., 2002;Zimmermann et al., 2002!. To determine if there is anadvantage to using these optimized or unconventional FPpairs over the classic ECFP:EYFP combination, we exam-ined the fluorescence properties of several FPs that are canbe used as FRET donors and acceptors ~Table 1!. Our setincludes cyan, green, and yellow FPs derived from theAequorea victoria green FP, as well as two recent additionsto the commercially available set of FPs, copGFP and phiYFP

~Shagin et al., 2004!. Because a true monomeric nature ishighly desirable for FP FRET experiments, we introducedthe A206K monomeric mutation ~Zacharias et al., 2002!into some of the Aequorea-derived FPs. This mutation didnot have an effect on the extinction coefficient and quan-tum yield of EGFP and Cerulean. In contrast, mutation ofthe yellow variants, Citrene and Venus, resulted in a de-creased quantum yield and an increased extinction coeffi-cient. These effects were similar in magnitude to the effecton the EYFP~Q69K! variant that was reported by Zachariaset al. ~2002!, and did not affect the overall brightness. TheFörster distances for various donor:acceptor pairings wasalso calculated ~Table 2!. A large increase in the Försterdistance was observed by exchanging proteins with a lowquantum yield donor ~ECFP! for a high quantum yielddonor ~mCerulean!. In contrast, a much smaller increase isseen by exchanging EYFP for a more recent generationmCitrene or mVenus ~Fig. 1!. The greatest Förster distancesoccur for the green to yellow FP combinations, with amaximum of 5.84 nm for the mEGFP:phiYFPm pairing.

To test whether the theoretical advantages of usingoptimized and nontraditional FP FRET pairs translated toan experimental advantage, two sets of tandem donor:accep-tor pairs with different FRET efficiencies were made foreach FP FRET pairing. A short linker consisting of 10amino acids was used to generate pairs with high FRETefficiency, and a long 44-amino-acid linker was used togenerate pairs with lower FRET efficiency. These coupled

Table 1. Properties of FPs*

Single-component fit Two-component fitFluorescentprotein «~f! t1 ~SD! x2 t1 ~SD! rel. % t2 ~SD! rel. % x2

ECFP 29,000 ~0.37!1 3 ~0.005! 4 3.6 ~0.033! 86 1.2 ~0.043! 14 1.2a

Cerulean 43,000 ~0.62!1 3.42 ~0.0083! 1.44a

mCerulean 44,000 ~0.62! 3.40 ~0.0110! 1.44a

EGFP 55,000 ~0.60!2 2.93 ~0.0066! 1.19a

mEGFP 55,000 ~0.62! 2.93 ~0.0066! 1.21a

copGFP 70,000 ~0.60!3 2.74 ~0.0061! 1.24a

EYFP 84,000 ~0.61!2

Citrene 77,000 ~0.76!4

mCitrene 90,000 ~0.62! 3.67 ~0.0089! 1.30a

Venus 92,000 ~0.57!5

mVenus 96,000 ~0.52! 3.37 ~0.0078! 1.29a

phiYFP 115,000 ~0.60!3 2.82 ~0.0037! 3.34 3.15 ~0.029! 90.5 1.22 ~0.063! 9.5 1.61a

phiYFPm 107,000 ~0.22! 3.69 ~0.0101! 1.29a

*Spectroscopic values were determined from recombinant protein purified to greater than 95% homogeneity. Absorbance and emissionwavelengths are expressed in nm and extinction coefficients ~«! is expressed in M�1cm�1. Quantum yields ~f! were calculated asdescribed in the Material and Methods section. Fluorescence lifetime values were determined from recombinant protein purified togreater than 95% homogeneity using time-correlated single photon counting spectroscopy. Relative % is the normalized relativecontribution of the fluorescent species characterized by its fluorescence lifetime constant t. Error is reported as standard deviation ofthe fit ~SD! and goodness of fit is reported by x2.aIncreased components did not significantly improve fit according to the conventional x2/2 criteria. 1Rizzo et al. ~2004!. 2Pattersonet al. ~2001!. 3Shagin et al. ~2004!. 4Griesbeck et al. ~2001!. 5Nagai et al. ~2002!.

Comparison of GFP FRET Pairs 243

FRET pairs were then recombinantly expressed and puri-fied, and the FRET efficiency of each tandem pairing wascalculated from time-resolved fluorescence lifetime measure-ment of donor fluorescence ~Table 3!. FRET efficienciesobserved for pairs containing two Aequorea-derived FPswere consistent with linker length, with shorter linkersgenerating increased FRET efficiencies. However, tandempairs containing either copGFP or phiYFP formed aggre-gates in solution and produced a variety of unexpectedeffects on their fluorescence properties. In the C-terminalposition, copGFP developed a second fluorescence lifetimecomponent. Fusion of phiYFP with a second energy transferpair disrupted maturation of fluorescence in either position~data not shown!. Site-directed mutagenesis was also used

to modify phiYFP to facilitate the generation of N-terminalfusions. This second generation phiYFP ~phiYFPm! wasfound to be less bright than phiYFP and did not toleratefusion to a donor FP, as indicated by the absence of energytransfer from mCerulean ~Table 3!. Among the Aequorea-derived FPs, the greatest FRET efficiency was observedbetween mCerulean and mVenus. Contrary to predictionsfrom the Förster distance calculations, we found FRETbetween green and yellow FPs to be less efficient. How-ever, it is unclear if this is a result of subtle differences inchromophore orientations of green and cyan FPs or fromcontamination of a lifetime component from direct excita-tion of the yellow FP acceptor.

Optimized Donor Excitation

FRET measurements are typically made using excitationparameters that favor the donor. In the case of the FPs, theyellow FP acceptor is brighter than the cyan FP donor, andtherefore minimization of direct excitation of the acceptoris required to enhance the quantification of FRET. To deter-mine the excitation wavelength that minimizes direct excita-tion of the acceptor and maximizes donor excitation, theFRET ratio of acceptor to donor fluorescence was deter-mined for coupled FP pairings with short or long linkers~Fig. 2B!. The difference in the FRET ratio was then plottedas a function of excitation wavelength. Using this method,the optimized excitation wavelength was determined fromthe plot maxima ~Fig. 2C!. The optimized wavelength for acyan:yellow FP pairing is approximately 425 nm. Thisparticular wavelength is not found on most commercialconfocal systems, and its use requires extensive optical cor-rections ~van Rheenen et al., 2004!. However, use of atunable Ti:Sapphire laser does allow optimized two-photonexcitation. To determine optimized two-photon excitationwavelengths, we measured two-photon cross sections foreach of the FPs ~Fig. 3!. Optimized two-photon excitationwavelengths were then calculated for each of the Aequorea-derived FP pairs ~Table 3!. We were unable to measure

Table 2. Calculated Förster Distances ~in Nanometers! for FP Pairings*

Acceptor

Donor EYFP mVenus mCitrene phiYFPm mEGFP copGFP mCerulean

ECFP 4.89 4.95mCerulean 5.33 5.40 5.36 5.49 5.03 4.99 3.52mEGFP 5.71 5.65 5.84 4.53 3.96 1.69copGFP 5.61 5.57 5.72 4.80 4.34 1.95phiYFPm 3.91 3.83 4.46 2.27 1.87 0.83mCitrene 5.15 5.05 5.67 3.09 2.51 1.33mVenus 4.95 4.85 5.48 2.84 2.20 1.09

*Förster distances were calculated as previously described ~Patterson et al., 2000!. Overlap integrals were calculated from spectra ofpurified protein as described in the Materials and Methods section.

Figure 1. Theoretical changes in FRET efficiency due to exchangeof EYFP and ECFP for optimized variants. Plot of FRET efficiency~E ! versus distance separating the FRET pair, where E � r0

6 /~r0

6 � r 6! and r is the distance separating the FRET pair and r0 isthe Förster distance reported in Table 2 for each donor:acceptorpair: ECFP:EYFP ~black!, ECFP:mVenus ~red!, mCerulean:EYFP~green!, and mCerulean:mVenus ~blue!.

244 M.A. Rizzo et al.

two-photon cross sections of copGFP, phiYFP, and phiYFPmas a result of extensive aggregation of these proteins insolution, which prevented accurate quantification of pro-tein concentrations by fluorescence correlation spectros-copy. The effect of wavelength optimization on crosstalkacceptor excitation is shown in Figure 4. Cells expressingmVenus or mCerulean were mixed prior to seeding andimaged using a conventional 458-nm Ar laser ~Fig. 4A! oroptimized 820-nm two-photon excitation ~Fig. 4B!. Excita-tion with the optimized wavelength greatly reduced cross-talk excitation of mVenus.

Comparison of FRET Imaging Methods

Numerous approaches have been proposed for imaging FPFRET ~Jares-Erijman & Jovin, 2003!. Ratiometric detection

of changes in acceptor to donor fluorescence is perhaps thesimplest method for quantifying changes in intramolecularFRET. This method has the benefit of high contrast, and it isparticularly well suited for intramolecular FRET measure-ments, because the stoichiometry of the FRET pairs isknown and crosstalk between fluorescence channels is con-sistent. To improve quantification of FRET, methods forminimizing excitation crosstalk and spectral separation offluorescence emission have been proposed. To quantify thebenefit of reducing crosstalk, we expressed conjoinedmCerulean:mVenus pairs in COS7 cells and measured FRETusing either conventional or optimized excitation. Donorand acceptor fluorescence were separated using either afilter bandpass strategy or spectral separation by linearunmixing prior to ratioing ~Fig. 5!. To account for theimpact of image processing on the overall quality of the

Table 3. Properties of Conjoined FP FRET Pairs*

Single component fit Two component fitDonor:Acceptorlinker t ~SD! x2 t1 ~SD! rel. % t2 ~SD! rel. % x2 E

opt. ex~2-photon!

ECFP:EYFP 428 ~820!Short 2.64 ~0.0038! 4.04 3.24 ~0.0234! 82.7 1.10 ~0.0255! 17.3 1.16a 0.120Long 2.81 ~0.0035! 4.37 3.33 ~0.0185! 86.6 1.06 ~0.0243! 13.4 1.16a 0.063

mCerulean:mVenus 427 ~820!Short 2.77 ~0.0115! 1.24a 0.185Long 2.99 ~0.0112! 1.26a 0.121

mCerulean:mCitrene 427 ~820!Short 2.89 ~0.0114! 1.26a 0.150Long 3.01 ~0.0106! 1.33a 0.115

mCerulean:phiYFPm 427Short 3.35 ~0.0117! 1.33a 0.015Long 3.37 ~0.0114! 1.27a 0.009

mCerulean:mEGFP 427 ~840!Short 2.98 ~0.0108! 1.31a 0.124Long 3.11 ~0.0107! 1.25a 0.085

mCerulean:copGFP 425Short 2.59 ~0.0093! 1.33a 0.238Long 2.94 ~0.0097! 1.26a 0.135

mEGFP:mVenus 466 ~840!Short 2.44 ~0.0077! 1.30a 0.167Long 2.56 ~0.0068! 1.25a 0.126

mEGFP:mCitrene 465 ~840!Short 2.50 ~0.0077! 1.34a 0.147Long 2.60 ~0.0069! 1.33a 0.113

copGFP:mVenus 467Short 1.52 ~0.0066! 4.63 2.47 ~0.0395! 60.1 0.69 ~0.0159! 39.9 1.11a 0.442Long 1.60 ~0.0042! 3.94 2.10 ~0.0245! 67.7 0.81 ~0.0215! 32.3 1.09a 0.416

copGFP:mCitrene 466Short 2.33 ~0.0070! 3.74 2.84 ~0.0250! 84.6 0.72 ~0.0253! 15.4 1.10a 0.150Long 1.74 ~0.0041! 4.32 2.32 ~0.0298! 66.9 0.92 ~0.0233! 33.1 1.20a 0.365

*Donor fluorescence lifetime values were determined from recombinant protein using time-correlated single-photon counting spectroscopy. Relative % isthe normalized relative contribution of the fluorescent species characterized by its fluorescence lifetime constant t. Error is reported as standard deviationof the fit ~SD! and goodness of fit is reported by x2.aIncreased components did not significantly improve fit according to the conventional x2/2 criteria. FRET efficiency ~E ! was calculated from time constantsgenerated for a single component fit and fluorescence lifetimes of the donor alone as reported in Table 1. Optimized excitation wavelengths weredetermined for single-photon and two-photon excitation as described in the Materials and Methods section.

Comparison of GFP FRET Pairs 245

data, the difference between short and longer linker FRETpairs was calculated and compared to the standard devia-tion of the short linker pair as a measure of the effective

dynamic range ~Table 4!. Thus, a superior method is onethat maximizes a statistically meaningful contrast resultingfrom a change in FRET.

The overall FRET ratios for the short and long linkerpairs were increased by crosstalk mVenus excitation, yet thenet increase to the FRET ratio remained unchanged regard-less of cross-talk reduction by either optimized excitation orlinear unmixing. However, the relative change to the FRETratio increased from a 1.4–fold increase using 458-nm exci-

Figure 2. Determination of optimized excitation wavelengths.A: Excitation spectra of ECFP ~solid line! and EYFP ~dotted line!.B: Ratio of acceptor ~525 nm! to donor ~475 nm! fluorescence ofECFP:EYFP conjoined pairs separated by a short linker ~solid line!and long linker ~dotted line! versus excitation wavelength. C: Plotof ECFP excitation spectra divided by the difference in the FRETratio of the short- and long-linker ECFP:EYFP pairs indicates theoptimized wavelength for the ECFP:EYFP FRET pair at the peak ofthe plot.

Figure 3. Two-photon cross sections ~fFs2P! of mCerulean ~A!mEGFP ~B! and mCitrene ~C, closed circles! and mVenus ~C, opencircles!.

246 M.A. Rizzo et al.

tation and filtered collection to a 1.9–fold increase usingoptimized excitation and linear unmixing. Although thischange does favor the application of linear unmixing toFRET-based imaging, it is largely offset by the effects ofimage processing ~see dFRET/SD; Table 4!.

Corrective algorithms are most appropriately appliedto intermolecular FRET experiments, and many solutionshave been found that produce reliable correction ~Berney &Danuser, 2003!. However, these methods are not routinelyapplied to FRET measurement, due in large part to theircomplicated and time-consuming collection strategy. Threemethods we tested were all able to reliably distinguish thepresence of FRET from cells expressing unlinked mCeruleanand mVenus ~data not shown!. However, when applied toanalysis of our short and long linker FRET pairs, we found alarge amount of variance in the data set, which diminishedthe overall usefulness of the correction ~Table 4!.

Figure 4. Increased dynamic range using spectral imaging withoptimized wavelengths. COS7 cells were electroporated to intro-duce either mVenus-C1 or mCerulean-N3 plasmids and mixedprior to seeding on coverslips. Spectral imaging and linear unmix-ing were used to separate mCerulean ~cyan! and mVenus ~yellow!fluorescence following excitation at 458 nm ~A! or 820 nm ~B!.

Figure 5. FRET ratio images of mVenus/mCerulean fluorescence from cells expressing linked mCerulean:mVenus.Spectral images were collected using either 458-nm excitation or 820-nm two-photon excitation. Separation of mVenusand mCerulean channels was performed by filter-type extraction of mCerulean ~472–482 nm! and mVenus channels~525–536 nm! or by linear unmixing using reference spectra from cells expressing unlinked mCerulean or mVenus.

Figure 6. FLIM images of mCerulean fluorescence ~820-nm excitation, 460–500 nm collection!. COS7 cells weretransfected with plasmids encoding mCerulean ~A!, mVenus:mCerulean ~B! conjugates separated by the long linker, orshort linker ~C! or unlinked mVenus and mCerulean ~D!. t values were averaged from at least 15 cells.

Comparison of GFP FRET Pairs 247

Techniques such as donor dequenching or FLIM areused to directly determine FRET efficiency. We found thatdirect measurement of the donor lifetime by FLIM ~Fig. 6!was both a reliable and accurate and method for deter-mining FRET efficiencies ~E � 0.12 for the long-linkermCerulean:mVenus pair and E � 0.19 for the shorter linkerpair!. Fluorescence lifetimes were shorter in cells ~comparelifetimes in Fig. 6 to Table 1! than in cuvettes, but the energytransfer efficiencies were similar. Although shortening ofthe observed in vitro lifetime has been consistently reportedin cells ~Pepperkok et al., 1999; Hanley et al., 2001!, themechanism is unclear. The drawback to FLIM is the require-ment for specialized instrumentation and long acquisitiontimes that are not always suitable for living cell experiments.More commonly, photo-destruction of the acceptor fluoro-phore is used to determine the presence of FRET. Becausethe amount of donor dequenching resulting from acceptorbleaching is directly related to the FRET efficiency, thismethod has been widely used as a simple method fordetecting FRET and quantifying FRET efficiency. However,we found that donor dequenching reliably and consistentlyreported greater FRET efficiencies ~Table 4! than the othermethods. We observed this to be true for over 200 cellsexpressing various combinations of yellow and cyan FPswith different linkers. Moreover, for ECFP:EYFP pairs, FRETefficiencies determined by donor dequenching were as muchas twofold greater than efficiencies for purified protein

determined by fluorescence lifetime and sensitized emissionby the method of Clegg ~1992!.

We examined three possibilities that could result in anincreased FRET efficiency. First, the possibility of increasedFRET in these preparations was ruled out. Fluorescenceemission spectra of recombinantly purified protein wascompared to spectra from cellular expression of the samepairing ~Fig. 7A!. The fluorescence emission spectra of thecellular preparations were indistinguishable from the recom-binant protein preparation. In addition, FRET efficienciesobtained from time-resolved fluorescence lifetime measure-ments of purified protein were equivalent to FRET efficien-cies calculated from FLIM of cells expressing short- andlong-linker mCerulean:mVenus pairs. These results indicatethat conjoined mCerulean:mVenus pairs exhibit an equiva-lent amount of FRET whether expressed in cells or recom-binantly expressed and purified. Second, we investigatedwhether the additional dequenching could be accounted forby photoconversion of mVenus during the photobleachingperiod, because photoconversion of other Aequorea FPs hasbeen described ~Patterson et al., 1997; Creemers et al.,1999!. However, we found no evidence of photoconversionof mVenus to a species with mCerulean-like fluorescence asa result of the bleaching period ~black line, Fig. 7F,G!.

Another source for the extra FRET observed in ourdequenching experiments was suggested by our observationthat different excitation parameters yield different donor

Table 4. Comparison of FRET Detection Methods*

mCerulean:mVenusE � 0.12

mCerulean:mVenusE � 0.19

Technique Value SD Value SD 6dFRET6/SD

RatiometricFilters

458-nm excitation 2.58 0.29 3.60 0.57 3.55820-nm excitation 1.62 0.18 2.39 0.41 4.33

Linear unmixing458-nm excitation 1.77 0.28 2.66 0.53 3.14820-nm excitation 1.05 0.21 1.98 0.53 4.51

Corrective processingFRETN1 0.00008 0.00007 0.00023 0.00023 2.18Fc2 0.024 0.028 0.030 0.036 0.21NFRET

3 0.086 0.047 0.139 0.079 1.14Donor dequenching

458-nm excitation 0.23 0.046 0.31 0.055 1.89800-nm excitation 0.21 0.053 0.27 0.046 1.00

FLIMt ~ns! 2.40 0.16 2.20 0.25 1.30

A-FRET800-nm excitation 0.225 0.023 0.199 0.03 0.86

*COS7 cells expressing mCerulean:mVenus short- and long-linker pairs were imaged using indicated technique. Values were from atleast 20 cells. Dynamic range is indicated by the absolute value of the difference in short- and long-linker FRET divided by the SD ofshort-linker FRET. 1Gordon et al. ~1998!. 2Youvan et al. ~1997!. 3Xia and Liu ~2001!.

248 M.A. Rizzo et al.

Figure 7. A: Spectral imaging ~800-nm excitation! of long-linker mVenus:mCerulean in cells ~red! and purified, recombinant protein insolution ~black!. Spectra were normalized for total fluorescence ~n � 10!. B: Ratio of mVenus/mCerulean single-photon excitation spectra~black, bottom scale! and two-photon cross sections ~red circles, top scale!. C: COS7 cells were cotransfected with plasmids encodingmVenus ~green! and mCerulean ~blue! prior to spectral imaging and separation by linear unmixing. Spectral imaging was performed byalternating 458-nm excitation ~D,F! and 800-nm excitation ~E,G!. D and E show emission spectra of cells ~blue–cell 1; green–cell 2;orange–cell 3; red–cell 4; black–cell 5! prior to bleaching. F,G: mCerulean fluorescence of cells in C before ~0–50 s! and after ~300 s!bleaching of mVenus fluorescence using a 514-nm laser line.

Comparison of GFP FRET Pairs 249

dequenching ~Table 4!. Taken together with the fact thatmVenus is highly absorbent compared to mCerulean, thisresult suggested to us that an inner filter effect resultingfrom absorption of excitation energy by mVenus could be asource of donor dequenching in addition to FRET. Thus,bleaching of mVenus would result in an increase in thequantity of donor absorption independent from FRETdequenching. Examination of the fraction of mVenus/mCerulean single- and two-photon absorption agrees withthis hypothesis ~Fig. 7B! because the magnitude of “extra”donor dequenching in short- and long-linker pairs is equiv-alent to the fraction of mVenus/mCerulean absorption atboth 458 nm and 800 nm ~Table 4!. To test the effect ofmVenus absorption on measurement of mCerulean fluores-cence, cells were cotransfected with mVenus and mCerulean.

No FRET was observed in these preparations as determinedby FLIM ~Fig. 6D! or A-FRET ~Fig. 8D,E!; however, photo-bleaching of mVenus resulted in increased mCerulean fluo-rescence in cells expressing a great amount of mVenus~Fig. 7F!. This effect was proportional to the amount ofexpression. Reduction of mVenus absorption by switchingto an optimized two-photon excitation wavelength dimin-ished the mVenus inner filter effect but did not entirelyeliminate it ~Fig. 7G!. These results indicate that the de-quenching observed in these cells is both concentration andwavelength dependent. Because FRET efficiency is not wave-length dependent, and because two other measures did notdetect the presence of FRET, we conclude that the source ofextra dequenching in our FRET experiments is due toreduction of the mVenus inner filter effect. Thus, quantifi-

Figure 8. Measurement of FRET by fluorescence polarization microscopy. A: Steady-state anisotropy ~r! spectra weremeasured ~425-nm excitation! for solutions containing mCerulean ~black! and mCerulean:mVenus conjoined pairscontaining the short ~red! and long ~blue! linkers. B: Excitation scan of mVenus anisotropy ~525 nm! for mCerulean:mVenus conjoined pairs containing the short ~red! and long ~blue! linkers. C: Steady-state anisotropy ~r! spectra weremeasured using two-photon excitation ~800-nm excitation! for solutions containing mCerulean ~black! and mCerulean:mVenus conjoined pairs containing the short ~red! and long ~blue! linkers. D: Scatter plot of sensitized emissionanisotropy data. Each point represents the sensitized emission of anisotropy calculated for an individual COS7cell expressing mCerulean, unlinked mVenus and mCerulean, or mVenus:mCerulean with short and long linkers.E: Anisotropy images ~pseudocolored! of COS7 cells expressing mCerulean, unlinked mVenus and mCerulean, ormVenus:mCerulean with short and long linkers. Anisotropy images were calculated from sensitized emission imagestaken at parallel and perpendicular polarizations using vertically polarized two-photon excitation ~800 nm!.

250 M.A. Rizzo et al.

cation of mCerulean:mVenus FRET efficiency solely by do-nor dequenching can potentially provide a false positiveindication of FRET.

An alternative approach for the detection of FRET isthe A-FRET technique ~Rizzo & Piston, 2005!, which utilizesfluorescence polarization microscopy ~Axelrod, 1979! tomeasure the anisotropy of the FRET acceptor. The large sizeand slow rotational diffusion of FPs result in highly polar-ized fluorescence emission ~r � 0.296 for mCerulean;Fig. 8A!. During FRET, donor fluorescence becomes morepolarized ~r � 0.342 for short linker, r � 0.337 with longlinker!, whereas acceptor fluorescence becomes highly depo-larized ~r � 0.0938 for short linker, r � 0.114 with longlinker!. Similar to other FRET detection methods, opti-mized excitation greatly enhances detection of desensitizedacceptor fluorescence ~Fig. 8B!. Contrast is further in-creased in the case of two photon excitation ~Fig. 8C!,because polarization selection is enhanced by two-photonabsorption ~Lakowicz et al., 1992!. We observed slightlygreater anisotropy values observed in cells, which may bedue to immobilization of proteins in the cytoplasm. A-FRETreliably distinguished between FRET ~Fig. 8D! between linkedFPs and cotransfected cells ~Fig. 8E! and provided thegreatest contrast between linked and unlinked pairs.

DISCUSSION

The Aequorea-derived cyan and yellow FPs are currently thebest characterized and most reliable variants for use inFRET experiments. In addition, the extensive developmentof this line of FPs has produced variants that are bothbright and essentially monomeric, making them exceptionalchoices for use in FRET assays. Other FPs are currentlybeing developed with properties that would also be goodchoices for FRET assays. For example a copGFP:phiYFPpairing has a greater Förster distance than the mCerulean:mVenus pair along with a greater spectral separation thanthe mEGFP:mVenus pair. However, the biochemical proper-ties, particularly spurious formation of aggregates, of copGFPand phiYFP are not yet suitable for the reliable generationof fusion proteins that retain the fluorescent properties oftheir respective monomers. Thus, further development isneeded to make them more suitable for FRET assays andthis study provides a good methodology for evaluatingpotential FRET pairings.

Considering FPs as FRET donors, we observed the bestresults using mCerulean. Because its quantum yield is 50%greater than ECFP, there is a large increase in the Försterdistance for mCerulean FRET as compared to ECFP. Further-more, FRET to mVenus was more efficient than FRET tomCitrine, which is consistent with the greater extinctioncoefficient of mVenus. Based on Förster distance calcula-tions, FRET to a yellow FP is even more efficient frommEGFP than from mCerulean; however, this was not ob-

served experimentally. Our observations suggest that thelinkers conjoining our FPs do not allow freely flexible mo-tion. The linkers are small relative to the FPs and werearbitrarily designed with no specific attention to flexibility.Together with the slow rotational diffusion of FPs, FRETin our conjoined pairs is likely to be very sensitive tomolecular differences in the orientation of the mEGFP andmCerulean dipoles. Although this limits the usefulness ofthe conjoined pairing strategy for comparing different spe-cies of FPs, it is a powerful tool for analysis of multiplegenerations of a single FPs, such as a comparison betweenEYFP, Citrene, and Venus.

The extensive spectral overlap between green andyellow FPs makes changes to the overall spectra more diffi-cult to detect than for the mCerulean donor, especiallybecause the optimized wavelength for a mEGFP:yellow FPpairing is 20 nm below the peak excitation of mEGFP.For mCerulean:yellow FP pairings, the optimized excitationwavelength is within 10 nm of the mCerulean peak. As aresult, optimized excitation of mCerulean:yellow FP pair-ings provides better contrast than for mEGFP:yellow FPpairings.

The FRET properties measured here between cyan andyellow FPs all closely follow Förster theory. This is in con-trast to the cyan and yellow FPs developed by Nguyen andDaugherty ~2005!, who found fourfold greater contrast fortheir CyPet:YPet FRET-based sensor than a sensor contain-ing ECFP and Venus, despite reductions in the donor quan-tum yield and acceptor extinction coefficient. Because thisobservation is not consistent with Förster theory, it is likelyadditional factors affect the overall contrast of the CyPet:YPet FRET-based sensor that are unrelated to FRET, such asdegradation of the acceptor following cleavage.

A method for calculating the optimized wavelength forFRET pairings was previously presented ~van Rheenen et al.,2004!. An optimized wavelength of 432 nm for ECFP:EYFPFRET was determined by following changes in the ratio ofEYFP to ECFP fluorescence of a FRET-based calcium sensorand multiplying this ratio change by the ECFP excitationspectra. This approach may be affected by conformationalchanges in the particular biosensing domain, which, inturn, have an impact on the FRET pairing. Here we presentan alternative method to calculate optimized wavelengthsfor each of our FRET pairings that is free of secondarybiosensor complications ~Table 3!. A limitation of our cal-culation is that it is based on a 1:1 stoichiometry for donorand accepter. For cases where the donor:acceptor stoichiom-etry is unknown, it may be useful to use a more blue-shiftedwavelength in the case of excess acceptor or a red-shiftedwavelength that is closer to peak donor excitation in thecase of excess donor.

Many corrective algorithms have been derived to re-duce crosstalk in microscopy-based applications. The threemethods of corrective processing used in this study ~Youvanet al., 1997; Gordon et al., 1998; Xia & Liu, 2001! were allable to reliably discriminate FRET from non-FRET condi-

Comparison of GFP FRET Pairs 251

tions. However, application of a corrective algorithm pro-vided less contrast between short- and long-linker FRETpairs than straight ratioing. This was not unexpected, be-cause laser scanning images contain a great amount ofnoise, which tends to be amplified during corrective process-ing. Thus, while corrective processing algorithms can cer-tainly enhance discrimination of FRET and certain types ofFRET quantification, application of this technique is notbeneficial to all types of FRET imaging. For intramolecularFRET experiments with an experimentally defined FRETpair ratio, straight ratiometric imaging provides better con-trast. This is particularly true if crosstalk is minimizedthrough the use of optimized donor excitation conditionsalong with spectral imaging techniques. In this case, weobserved a greater than 75% increase in the FRET ratio byshortening the linker of our coupled dimer. In contrast,deriving the FRET efficiency by measuring the fluorescencelifetime of the donor was reported as only a 35% decrease~Table 4!.

Due to the potential for false positive indication ofFRET from direct acceptor excitation, uncorrected ratiomet-ric techniques are not reliable indicators of intermolecularFRET. More commonly, methods that directly measure FRETefficiency, such as FLIM and donor dequenching, have beenused to directly detect the presence of intermolecular FRET.We found FLIM to be very accurate and reliable. However,this technique requires extensive instrumentation that is notyet as commonplace as standard confocal and wide-fieldmicroscopy, and it often requires long integration times thatmake it less adaptable to living-cell applications. Somerecent advances in FLIM instrumentation ~Krishnan et al.,2003; Agronskaia et al., 2004! gives considerable improve-ment to the overall collection speed, but these solutions arestill in development, will ultimately be somewhat expensive,and thus are not widely accessible. Consequently, donordequenching has been more widely used as a standardmethod, despite the drawback of sample destruction. Here,we have identified another important drawback that mayresult in false positive indication of FRET. Although FRET isa source of donor dequenching, our identification of arelevant inner filter effect as an additional source of de-quenching makes this assay far less attractive as an indicatorof CFP:YFP FRET. This is due, in part, to the fact that theoverall effect is proportional to the amount of acceptorexpression and thus is difficult to quantify and correct forin a typical transient expression system. Fortunately, thereare special circumstances that limit the impact of the innerfilter effect, such as the case of two-membrane delimitedprobes. Alternatively, A-FRET is a simple and high contrastmethod for determining the presence of FRET. Becauseanisotropy is typically measured to an accuracy of greaterthan 0.01 in a microscope, this approach offers a far greaterdynamic range than even FLIM ~Rizzo & Piston, 2005!. Thesimplicity of A-FRET measurements also makes it wellsuited for high-throughput screening purposes where thedesired readout is FRET or no FRET. However, it is impor-

tant to note that this technique also has some very impor-tant limitations. First, this technique requires a very highinitial anisotropy of both the donor and acceptor, restrict-ing its use to FP experiments. Second, this technique doesnot discriminate different FRET efficiencies with very muchcontrast. Thus for intramolecular FRET experiments, ratio-metric methods produce the best contrast.

SUMMARY

In conclusion, we find that use of monomeric Cerulan andVenus FPs in FRET experiments is a more efficient andadvantageous pairing to use for FRET experiments thanother currently available pairings. In addition, ratiometricimaging provides the best contrast for intramolecular FRETexperiments, whereas A-FRET provides the best discrimina-tion of intermolecular FRET.

ACKNOWLEDGMENTS

Funding for this work was provided by the U.S. NationalInstitutes of Health grants DK60275, and DK067415~M.A.R.!, and DK53434, CA86283, GM072048 ~to D.W.P.!,the U.S. National Science Foundation grant BBI-9871063~to D.W.P.!, and the U.S. Department of Defense MedicalFree-Electron Laser program grant F49620-01-1-0429 ~toD.W.P.!.

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