TripleFRET Measurements in Flow Cytometry

�Akos Fabian,1 Gabor Horvath,2 Gyorgy Vamosi,1 Gyorgy Vereb,1,3 Janos Szollo†si1,3*

� AbstractA frequently used method for viewing protein interactions and conformation, Forster (fluo-rescence) resonance energy transfer (FRET), has traditionally been restricted to two fluoro-phores. Lately, several methods have been introduced to expand FRET methods to threespecies. We present a method that allows the determination of FRET efficiency in three-dyesystems on a flow cytometer. TripleFRET accurately reproduces energy transfer efficiencyvalues measured in two-dye systems, and it can indicate the presence of trimeric complexes,which is not possible with conventional FRETmethods. We also discuss the interpretation ofenergy transfer values obtained with tripleFRET in relation to spatial distribution of labeledmolecules, specifically addressing the limitations of using total energy transfer to determinemolecular distance. ' 2013 International Society for Advancement of Cytometry

� Key termsForster (fluorescence) resonance energy transfer; tripleFRET; relay transfer; trimericcomplexes

FLUORESCENCE Resonance Energy Transfer (FRET), originally described by T. Forster

(1), is a nonradiative energy transfer process taking place between an excited donor fluor-

ophore and an acceptor dye. The rate of energy transfer is inversely proportional to the

sixth power of the separation between the donor and the acceptor. In the past several dec-

ades, this phenomenon was implemented in biological systems as a nanometer-scale ruler

to view molecular distances and association patterns (2–8). To date, a myriad of meth-

odologies have been developed to measure FRET efficiency, although most of these were

limited to examining the interaction of only two different proteins at a time (9–13).

The realization that a multitude of processes in cellular biology are not restricted

to the interaction of only two molecules has fueled efforts to expand traditional two-

dye FRETmeasurements to include more interaction partners. Recently, several arti-

cles investigated the possibility of measuring FRET between three distinct fluorescent

molecules (14–26). The major aims of these studies were to establish the theoretical

background for tripleFRET measurements and use the method in biologically rele-

vant experiments. The multiple FRET processes that can occur between three fluoro-

phores were thoroughly characterized by Watrob et al. (16) by using DNA-bound

dyes with known distances. An interesting observation was that the presence of an in-

termediate fluorophore sensitizes the FRET process and extends the detectable dis-

tance range by about 50%. The three-color FRET phenomenon was also used to

study single-molecule Holliday-junction dynamics with donor photobleaching

experiments by microscopy (22). Another study showed the applicability of fluores-

cent proteins (CFP, YFP, and mRFP) to three-fluorophore FRET in live cell imaging

(20). The approach was validated with cleavable protein-linked fluorescent proteins,

and afterwards the interaction of stimulated EGFR with adaptor proteins (Grb2 and

Cbl) present in signal transduction was analyzed using acceptor photobleaching and

intensity-based microscopic techniques. A similar approach was used for the deter-

mination of TRAF2 homotrimerization in flow cytometry (27). In a more recent

study, three-chromophore FRET was used to reliably discriminate between DNA

strands labeled with the same three fluorescent dyes but with different intrastrand

dye distances (23).

1Department of Biophysics and CellBiology, Research Center for MolecularMedicine, Medical and Health ScienceCenter, University of Debrecen, Hungary2Institute of Innate Immunity, UniversityHospitals, University of Bonn, Germany3MTA-DE Cell Biology and Signaling ResearchGroup, Research Center for MolecularMedicine, Medical and Health ScienceCenter, University of Debrecen, Hungary

Received 20 September 2012; RevisionReceived 18 December 2012; Accepted 23January 2013

Grant sponsor: Hungarian National Re-search Fund; Grant numbers: OTKA OTKANK 101337 and K75752; Grant sponsor:New Hungary Development Plan co-financed by the European Social Fund;Grant sponsor: European Regional De-velopment Fund; Grant numbers: TAMOP-4.2.2-08/1-2008-0019, TAMOP-4.2.1/B-09/1/KONV-2010-0007, and TAMOP-4.2.2. A-11/1/KONV-2012-0025; Grant sponsor:Baross Gabor Program; Grant number:REG_EA_09-1-2009-0010.

Additional Supporting Information may befound in the online version of this article.

�A. Fabian and G. Horvath authorscontributed equally to the article.

*Correspondence to: Janos Sz€ollo†si,Department of Biophysics and CellBiology, Faculty of Medicine, Medicaland Health Science Center, University ofDebrecen, P.O.Box 39, Nagyerdei krt. 98,H-4012 Debrecen, Hungary

Email: szollo@med.unideb.hu

Published online 8 March 2013 in WileyOnline Library (wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.22267

© 2013 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 83A: 375�385, 2013

Although these studies described the phenomenon prop-

erly and demonstrated their applicability to biological samples,

they had restrictions concerning specimen selection (for

instance using rigid DNA strands) (15,22–25,28) or simplifica-

tions in the analysis (neglecting possible transfer processes, thus

obtaining only semi-quantitative FRET values) (22,27). Simpli-

fications were made because three-dye systems are exceedingly

more complex than conventional two-dye systems, with spectral

bleed-through from multiple fluorophores often obscuring the

true FRET signal. Several workarounds were developed, either

simplifying the imaging of three molecules to two fluorophores

(29) or forgoing complications arising from simultaneous ima-

ging of dyes with sequential bleaching (30). However, these

methods either require extensive molecular biology work to pre-

pare adequate samples, or the imaging process is itself cumber-

some and does not allow for live-cell or high-throughput ima-

ging. A general, relatively easy-to-implement method applicable

to biological systems with variable molecular stoichiometries

(such as cell surface protein clusters) has not been presented.

In this study, we discuss mathematical algorithms to cal-

culate FRET efficiencies for three-dye systems on a cell-by-cell

basis for flow cytometry. The method does not require any

special instrumentation beyond a commercial flow cytometer

capable of excitation at three distinct wavelengths and acquisi-

tion of six intensity channels. Preparation of samples is fast, as

labeling with fluorescently tagged antibodies is sufficient for

the measurement. Furthermore, the FRET calculation does

not require separate measurement of unquenched donor

intensities, thereby minimizing artifacts introduced by inter-

sample (and cell-by-cell) variability.

MATERIALS AND METHODS

Cell Lines

Human gastric cell line N87 with high ErbB2 and major

histocompatibility complex class I expression level (31)

was obtained from the American Type Culture Collection

(Rockville, MD) and grown according to the manufacturer’s

specification (in RPMI containing 10% fetal bovine serum, 2

mM l-glutamine, and 0.25% gentamicin in 5% CO2 atmo-

sphere) to confluency. For flow cytometry, cells were harvested

by treatment with 0.05% trypsin and 0.02% ethylenediamine-

tetraacetic acid before antibody labeling.

Conjugation of Antibodies with Fluorescent Dyes

In our experiments, we used the following anti-erbB2 anti-

bodies: pertuzumab (a gift from Hoffman-La Roche, Grenzach-

Wyhlen, Germany); trastuzumab (purchased from Hoffman-La

Roche, Grenzach-Wyhlen, Germany); and H76.5 antibody (pre-

pared from the hybridoma cell line, a kind gift of Yosef Yarden).

Aliquots of antibodies (about 1 mg/ml concentration) were

conjugated with the succinimidyl ester derivative of Alexa Fluor

488 (dye A), Alexa Fluor 546 (dye B), or Alexa Fluor 647 (dye

C), Life Technologies, Carlsbad, CA, dyes according to the man-

ufacturer’s specifications. The labeling ratios were in the range

of 2–4, where concentration quenching does not have a signifi-

cant effect on the fluorescence quantum yield of the fluoro-

phores (32). Relevant excitation and emission spectra of these

fluorescent dyes are shown in Figure 1.

Labeling Cells with Fluorescent Antibodies

For flow cytometry, freshly harvested cells were washed twice

in ice-cold phosphate buffered saline (PBS; pH 7.4). The cell pellet

was suspended in PBS at a concentration of 2 3 107 cells/ml.

Then 25 ml of conjugated antibodies were added to 25 ml of cellsuspension and cells were incubated for 30 min on ice. The excess

of antibody was at least fivefold above saturating concentrations

during incubation. The same procedure was used for FRET sam-

ples, in which a mixture of donor- and acceptor-labeled antibo-

dies was added to the cell suspension. Labeled cells were washed

twice with cold PBS and fixed with 1% formaldehyde in PBS.

Instrumentation and Sample Measurement

For flow cytometric measurements, we used a FACSVan-

tage SE with DiVa option flow cytometer (Becton Dickinson,

Franklin Lakes, NJ) equipped with a 488-nm water-cooled Ar-

gon-ion laser, a 532-nm diode pumped solid-state laser and a

633-nm air-cooled HeNe laser. The fluorescence detection

channels (emission filters and laser wavelengths used for exci-

tation) for the three fluorophores are shown in Table 1.

Theory of FRET

FRET is a nonradiative energy transfer process, in which an

excited donor molecule excites an adequately oriented fluores-

cent acceptor molecule via a long distance dipole-dipole interac-

tion. Transfer efficiency, which is the probability that a donor ex-

citation quantum is transferred to an acceptor, is given as:

E ¼ ktransfer

ktransfer þ kother; ð1Þ

where ktransfer is the rate constant of energy transfer and kotheris the sum of the rate constants of all other de-excitation

Figure 1. Normalized excitation and emission spectra of donor

and acceptor fluorophores. The gray shaded area corresponds to

the overlap integral of the emission spectrum of dye A and excita-

tion spectrum of dye C.

ORIGINAL ARTICLE

376 TripleFRET

pathways. The distance dependence of the transfer efficiency is

described by the following equation:

E ¼ 1

1þ r6

R60

; ð2Þ

where r is the separation of the donor and acceptor and

R0 is the so called Forster distance characteristic for the

donor-acceptor pair. The Forster distance is defined as

the distance at which the transfer efficiency between do-

nor and acceptor is 50%. This distance dependence

makes FRET efficiency a sensitive indicator of intermole-

cular distance.

The presence of FRET results in the quenching of donor

intensity and a simultaneous increase in acceptor fluorescence.

Multiple methods of FRET calculation exist, some of which

are based on the measurement of fluorescence lifetime,

whereas others use quenching of the donor or sensitized emis-

sion of the acceptor (9).

Theory of TripleFRET

Although a three-fluorophore FRET system is seemingly

complicated, it can be easily described by two distinct

schemes as shown by Watrob et al. (16). If we denote the

fluorophores with increasing excitation wavelength (e.g.,

blue, green, and red) as A, B, and C, these schemes are as fol-

lows (see also Fig. 2.): (1) the so-called relay or two-step

FRET process, when the energy from donor A is channeled

to C via B, and (2) two competitive FRET processes from do-

nor A to acceptor B and from donor A to acceptor C. Natu-

rally, there is also a third scheme, which is a combination of

the previous two when all possible FRET processes are

allowed.

In the first case, the relay FRET efficiency (Erelay) can

be given by the product of the two individual FRET effi-

ciencies:

Erelay ¼ EAB � EBC ð3Þ

For the second case, since the two FRET processes are compet-

ing for the same donor A, their individual FRET efficiencies

(EAB and EAC) have to be corrected for this competition. For-

tunately, these competitive FRET efficiencies (E0AB and E0AC)can be calculated from the noncompetitive two-dye FRET effi-

ciencies:

E0AB

EAB¼ kAB

kAB þ kAC þ kother

kAB þ kother

kAB

¼ kAB þ kother þ kAC � kAC

kAB þ kAC þ kother¼ 1� E0

AC ð4Þ

E0AB ¼ EABð1� E0

ACÞ E0AC ¼ EACð1� E0

ABÞ ð5Þ

E0AB ¼ EAB

1� EAC

1� EABEACE0AC ¼ EAC

1� EAB

1� EABEAC; ð6Þ

In the third case, both relay and competitive FRET can occur,

and total FRET efficiency (Etotal) is the sum of relay and direct

FRET corrected for competition for donor A:

Etotal ¼ E0AB � EBC þ E0

AC : ð7Þ

Evaluation of Transfer Efficiency

All transfer efficiencies were calculated with the equation

set described in the Results section of this article, unless other-

wise indicated. To evaluate FRET data obtained with flow

cytometry, a much-improved version of the AFlex software,

ReFlex (free-ware, available at http://www.freewebs.com/cyto-

flex) was used with the equations entered in the equation edi-

tor of the program and applying manual gating (33) (for the

gating strategy please see the Supporting Information). Trans-

fer efficiency values are given as median values of transfer effi-

ciency histograms. Flow cytometric dotplots and histograms

were generated with ReFlex, three-dimensional transfer effi-

ciency scatter plots were created with Wolfram Mathematica 7

(Wolfram Research, Champaign, IL).

Figure 2. Schematic drawing of a three-dye system with possible

energy transfer routes. E0AB and E0AC values refer to transfer effi-

ciencies measured in the presence of competition between trans-

fer processes from A to B and A to C. Erelay 5 E0AB. . .EBC is thetransfer efficiency between A and C via B, Etotal 5 E0AC 1 Erelay is

the total probability of energy transfer from A to C via the direct

and indirect mechanisms.

Table 1. Laser excitation wavelengths and emission filters

of intensity channels

CHANNEL LASERWAVELENGTH (NM) FILTER

I1 488 530/30

I2 488 585/42

I3 488 675/20

I4 532 585/42

I5 532 650 LP

I6 633 650 LP

ORIGINAL ARTICLE

Cytometry Part A � 83A: 375�385, 2013 377

Determining Cross-Excitation, Spillover, and Alpha-

Factors

Each fluorophore used has its ‘‘native’’ detection channel

corresponding to its optimal excitation and detection wave-

lengths. In most channels, however, the emissions of two or all

three dyes are mixed. Cross-excitation and spillover factors (S12 S12) account for the emission of dyes in channels not native

for the given dye. S factors were measured on single-labeled

samples according to Eqs. (10)–(12). Samples labeled with

Alexa Fluor 488 were used to calculate S1 and S9; samples la-

beled with Alexa Fluor 546 to calculate S2, S4, S10, and S12; and

samples labeled with Alexa Fluor 647 to calculate S3, S5, and S7.

Alpha factors are scaling factors correcting for the differ-

ence in the fluorescence quantum yields and detection efficien-

cies of donor and acceptor fluorophores. The alpha factor can

be considered as a ‘‘currency exchange rate’’ to scale loss of do-

nor emission to gain of acceptor emission. To calculate the

alpha factor, the intensity of the same number of excited donor

and acceptor fluorophores has to be measured at given wave-

lengths. This is most easily done by labeling a cell-surface pro-

tein with donor- and acceptor-tagged antibodies in separate

samples. Alternatively, two different antibodies can be used that

do not compete with each other for binding and that recognize

epitopes far enough apart so that energy transfer does not occur

(34,35). Another possibility is to apply epitopes with known

distances and well characterized transfer efficiencies, and set the

value of alpha to yield the reference transfer efficiencies (34).

When using fluorescent proteins, donor-acceptor fusion pro-

teins can be constructed where the expression level of both

fluorophores are the same. By varying the length of the linker

region (36) or by using an iterative method yielding E and

alpha simultaneously (37), alpha can be calculated.

For our experiments, the average intensity of several

thousand cells singularly labeled with Alexa Fluor 488, Alexa

Fluor 546, or Alexa Fluor 647 was used to obtain average

intensities for the calculation of alpha factors. The effects of

errors of S and alpha factors on measured transfer efficiency

are shown in Section 6 of the Supporting Information.

RESULTS

TripleFRET Measurements in Flow Cytometry

To measure FRET efficiency on a cell-by-cell basis, both

donor quenching and sensitized emission had to be considered

and a corresponding set of equations has to be solved. Below,

we present an initial set of equations for the scenario, where

both relay and direct energy transfer from A to C take place

(equations and their solutions for the cases of relay transfer

only, direct transfer only and no direct or relay transfer can be

found in the Supporting Information section). Six independ-

ent emission intensities I1 – I6 can be identified and broken

down into terms due to direct excitation and sensitized emis-

sion of the dyes: I1: quenching of donor A (by acceptors B and

C), native intensity channel to detect dye A; I2: sensitized

emission (from donor A) and quenching of acceptor B (by

acceptor C), I3: sensitized emission of acceptor C (from donor

A and donor B excited through donor A), I4: quenching of do-

nor B (by acceptor C), native intensity channel to detect dye

B; I5: sensitized emission of acceptor C (from donor B); and

I6: native intensity channel to detect dye C.

I1ð488 nm ! 530=30BPÞ ¼ IAð1� E0AB � E0

ACÞI2ð488 nm ! 585=42BPÞ ¼ S1IAð1� E0

AB � E0ACÞ

þ aABIAE0ABð1� EBCÞ þ S4IBð1� EBCÞ

I3ð488 nm ! 675=20BPÞ ¼ aACIAðErelay þ E0ACÞ

þ S12IAE0ABð1� EBCÞ þ S9 IA ð1� E0

AB � E0ACÞ ð8Þ

þ S10IBð1� EBCÞ þ S7IC

I4ð532nm ! 585=42BPÞ ¼ IBð1� EBCÞI5ð532nm ! 585=650BPÞ ¼ S2IBð1� EBCÞ

þ aBCIBE0BC þ S3IC

I6ð633nm ! 650LPÞ ¼ IC

As we wanted to give a full representation of the system, equa-

tions include the competitive FRETefficiencies E0AB and E0AC for

FRET from A to B and from A to C, respectively. There are alto-

gether seven unknowns: three unperturbed intensities IA, IB, and

IC from the three dyes (that would be measured in the absence of

FRET) and four transfer efficiencies E0AB, E0AC, EBC, and Erelay.

Since in its present form the equation system is underdetermined

(with only six independent equations for seven variables), a fur-

ther equation is required for a solution. Therefore Eq. 3 is used

(in a slightly modified form to account for competition between

dyes B and C) for a seventh independent equation:

Erelay ¼ E0AB � EBC ð9Þ

The spillover and cross-excitation factors (S1 – S12) were cal-

culated from single-labeled samples in the following way

(where Iqp is the intensity in channel p of a sample labeled with

only dye q conjugated to antibodies):

S1 ¼IA2IA1

S9 ¼IA3IA1

ð10Þ

S2 ¼IB5IB4

S4 ¼IB2IB4

S10 ¼IB3IB4

S12 ¼IB3IB2

ð11Þ

S3 ¼IC5IC6

S5 ¼IC4IC5

S7 ¼IC3IC6

ð12Þ

The alpha-factors can be determined empirically by the fol-

lowing formulas (where LX is the labeling ratio of the antibody

with fluorophore X and eX is the molar extinction coefficient

of fluorophore X at the indicated wavelengths):

aAB ¼ IB2IB1

� LA

LB� e

A488

eC488aAB ¼ IC3

IA1� L

A

LC� e

A488

eC488aAB ¼ IC5

IB4� L

B

LC� e

B488

eC488ð13Þ

Upon substituting Eqs. (9)–(13), the equation set 8 gives very

complicated solutions for all unknowns (except for IC). In our

experiments S5 was reproducibly 0 and therefore it was omit-

ORIGINAL ARTICLE

378 TripleFRET

ted from calculations thereby simplifying the formulas (how-

ever, it was measured each time to verify the validity of such a

simplification). The solutions contain several terms, which

upon factorization make the equations look simpler and their

physical meaning more prominent. These terms are labeled as

IX1 2 IX5, and can be considered as donor quenching (IX1 and

IX4) and bleed-through corrected sensitized emission (IX2, IX3,

and IX5) intensities:

IX1 ¼ I1

IX2 ¼ I2 � S1I1 � S4I4

IX3 ¼ I3 � S12I2 � ðS9 � S1S12Þ I1S7I6 � S4IX5 ð14ÞIX4 ¼ I4

IX5 ¼ I5 � S2I4 � S3I6

Using the equations above, we arrive at the following solutions

for the equation set (8):

IA ¼ IX1 þIX2

aABþ IX3

aAC

IB ¼ IX5 þ aBCIX4aBC

ð15Þ

IC ¼ I6

E0AB ¼ aABIX2

ðaABIX3 þ aACIX2 þ aABaACIX1Þð1� EBCÞ

¼ aACIX2ðIX5 þ aBCIX4ÞaBCIX4½aACIX2 þ aABðIX3 þ aACIX1Þ�

ð16Þ

E0AC ¼ aABIX3ð1� EBCÞ � aACIX2EBC

ðaABIX3 þ aACIX2 þ aABaACIX1Þð1� EBCÞ

¼ aABaBCIX3IX4 � aACIX2IX5aBCIX4½aACIX2 þ aABðIX3 þ aACIX1Þ�

ð17Þ

EBC ¼ IX5

IX5 þ aBCIX4ð18Þ

The noncompetitive FRET efficiencies can also be calculated

using Eq. (6):

EAB ¼ IX2ðIX5 þ aBCIX4ÞIX2ðIX5 þ aBCIX4Þ þ aABaBCIX1IX4

ð19Þ

EAC ¼ aABaBCIX3IX4 � aACIX2IX5aABaBCIX4ðIX3 þ aACIX1Þ � aACIX2IX5

ð20Þ

Based on Eq. (3), Erelay can also be given with the factorized

terms:

Erelay ¼aACIX2IX5

aBCIX4½aACIX2 þ aABðIX3 þ aACIX1Þ�ð21Þ

After substituting into Eq. (7), Etotal (the total percentage of

the energy absorbed by dye A that is eventually passed on to

dye C) can be calculated as:

Etotal ¼aABIX3

aACIX2 þ aABðIX3 þ aACIX1Þð22Þ

Or in a more simplified form:

Etotal ¼IX3

aACIA: ð23Þ

Transfer Efficiencies of Two- and Three-Dye Systems

To test our calculation method, first N87 cells were la-

beled with different antibodies against the ErbB2 protein.

Samples were prepared as a three-dye system as well as a cor-

responding set of two-dye systems (Table 2). For validation

purposes and to demonstrate the applicability of our method

in three-dye systems, all samples were evaluated (in addition

to our own equations) using an intensity-based method pre-

viously developed and tested for two-dye systems (38; for a

detailed description of the equations used for FRET analysis,

please see the Supporting Information). The use of non com-

peting antibodies against different epitopes of the same pro-

tein ensured intramolecular binding and proximity of the

dyes (4,39). The results of these measurements are listed in

Table 2. All permutations of a two-dye system with the three

fluorophores resulted in measurable transfer efficiencies.

Analyzing FRET in two-dye systems according to the triple-

FRETmethod produced identical transfer efficiency values as

conventional two-dye intensity-based FRET analysis. FRET

analysis of the three-dye system with tripleFRET resulted in

transfer efficiency values very similar to the ones obtained in

two-dye systems. Correction for competition between the

two acceptors further increased the agreement with the

values from two-dye systems. At the same time, traditional

intensity-based FRET failed to reproduce the FRET values of

the two-dye systems in the triple-labeled sample. Specifically,

EAB was underestimated (7.9% instead of 13.4%) and EACoverestimated (11.4% instead of 4.9%). The addition of dye

B to the labeling scheme consisting of only dyes A and C sub-

stantially increased the total energy transferred from A to C,

providing evidence for a relay transfer process in our intra-

molecular model system.

To demonstrate the sensitivity of tripleFRET calcula-

tions and its power to dissect populations with different

protein association patterns in biological systems, we

mixed the samples together described in Table 3 in the

same tube. Then energy transfer was measured by flow

cytometry for the mixed sample. A representative dot plot

and transfer efficiency histograms are shown in Figure 3.

As can be seen on the transfer efficiency histograms, a sin-

gle measurement of transfer efficiency allows discrimina-

tion of three distinct populations. When only EAB is meas-

ured, Specimens 1 and 6, Specimens 3 and 4, as well as

Specimens 2, 5, and 7 cannot be separated from one

another. When only EAC is analyzed, Specimens 1 and 2,

Specimens 4 and 5 as well as Specimens 3, 6, and 7 show

near identical transfer efficiency distributions. Total FRET

ORIGINAL ARTICLE

Cytometry Part A � 83A: 375�385, 2013 379

efficiency is similar in Specimens 1 and 2 as well as in

Specimens 4, 5, 6, and 7. The simultaneous calculations of

several transfer efficiencies is needed for discrimination of

specimens that show similar transfer efficiency distribu-

tions when only one FRET value is measured. The evalua-

tion of all three FRET efficiencies allowed us to discrimi-

nate between seven differently labeled specimens in the

same sample. The calculated transfer efficiency values for

the identified specimens are displayed in Table 3. The

transfer efficiency values measured in such a fashion were

in good agreement with FRET efficiencies obtained from

the specimens measured individually (data not shown).

TripleFRET in Three-Dye Systems with Different

Spatial Distributions of Dyes

Lastly, we altered the labeling scheme (see Fig. 4) so that

the three dyes could not colocalize on the same protein due to

competition between antibodies. This way we either achieved

a dye configuration where the transfer process from A to B is

intermolecular (Sample 2) or dye B excited by energy transfer

Table 2. Measured transfer efficiencies (%) in various dye systems

LABELING SCHEME DYE A: ALEXA FLUOR 488 DYE B: ALEXA FLUOR 546 DYE C: ALEXA FLUOR 647

Double (AB) H76.5 Trastuzumab –

Double (AC) H76.5 – Pertuzumab

Double (BC) – Trastuzumab Pertuzumab

Triple H76.5 Trastuzumab Pertuzumab

EAB EAC EBC

Double (AB) Triple Double (AC) Triple Double (BC) Triple

E as two-dye 13.5 7.9 4.9 11.4 45.1 44.3

E0 13.5 12.9 4.9 4.0 45.1 44.4

E 13.4 13.4 4.9 4.6 N.A. N.A.

Erelay Double (AC) Triple

N.A. 5.7

Etotal 4.9 10.4

Measured EAB, EAC, and EBC median transfer efficiencies in three-dye and corresponding two-dye systems. E as two-dye: traditional

intensity-based FRET analysis used for two-dye systems (38); E0, E, Erelay, and Etotal are competitive, noncompetitive, relay, and total FRETefficiencies calculated with the tripleFRET method; N.A.: not applicable. For standard deviations of transfer efficiencies, please see Section

5 in the Supporting Information.

Table 3. Labeling schemes and energy transfer efficiency values (%) measured for individual specimens after mixing and a single data ac-

quisition

ALEXA FLUOR 488 ALEXA FLUOR 546 ALEXA FLUOR 647

Specimen 1 Pertuzumab Trastuzumab –

Specimen 2 H76.5 Trastuzumab –

Specimen 3 H76.5 – Pertuzumab

Specimen 4 Trastuzumab – Pertuzumab

Specimen 5 Trastuzumab H76.5 Pertuzumab

Specimen 6 Pertuzumab Trastuzumab H76.5

Specimen 7 H76.5 Trastuzumab Pertuzumab

FRET EFFICIENCY AB AC BC RELAY TOTAL

Specimen 1 24.2 0.0 0.0 0.0 0.0

Specimen 2 12.3 0.0 1.4 0.1 0.3

Specimen 3 –0.3 4.9 0.0 0.0 4.8

Specimen 4 0.0 9.9 0.0 0.6 9.9

Specimen 5 9.8 9.6 33.0 2.9 12.4

Specimen 6 21.2 2.8 30.5 6.6 10.4

Specimen 7 13.5 4.5 44.2 5.5 10.2

For standard deviations of transfer efficiencies, please see Section 5 in the Supporting Information.

ORIGINAL ARTICLE

380 TripleFRET

from dye A was not in close proximity of dye C (Sample 3),

causing relay FRET to become minimal (Fig. 4). Transfer effi-

ciency was calculated with different initial equation sets con-

sidering four scenarios: simultaneous relay and direct transfer

from A to C; only relay transfer without direct transfer; only

direct transfer without relay transfer; no relay or direct trans-

fer (please see the Supporting Information for the equations

used in the individual scenarios). Results and comparison

with two-dye, dominantly intramolecular FRET values are

summarized in Table 4. In the case of Sample 1, the scheme

supposing direct and relay transfer to dye C gave the best

approximation of energy transfer values from two-dye systems

without neglecting any transfer processes. The same was true

for Sample 2, where assuming only relay transfer neglected the

substantial direct transfer process from dye A to C and sup-

posing only direct-FRET underestimated energy transfer from

A to B. However, for Sample 3, analysis involving simultane-

ous direct and relay transfer failed to give results with a physi-

cal meaning, as A to C transfer was found to be negative. Cal-

culations with only relay transfer produced a relay-FRET value

that was higher than the total energy transfer from A to C.

Therefore, a scheme involving only direct transfer gave the

best results, with physically plausible results obtained for all

calculated transfer efficiencies. However, in this case a small

but relevant amount of relay transfer was neglected, since total

transfer was higher in the three-dye system than in a system

with only dyes A and C. As we shall later discuss, in Sample 3

several different spatial distributions are measured at the same

time, which causes calculations assuming a single, homoge-

nous distribution to become inaccurate.

DISCUSSION

FRET measurements have gained wide acceptance as a

means of following changes in molecular distance and asso-

ciation. However, the fact that FRET requires a close proxim-

ity of the donor and acceptor dyes has limited the dynamic

range of these measurements. Furthermore, the spectral crite-

ria of overlap between the emission spectra of donor and ex-

citation spectra of acceptor meant that only two fluorescently

labeled molecules were viewed at a time. Recent studies have

shown that, by adding a third dye, the dynamic range of

FRET can be extended via relay-FRET. With the addition of a

new dye, new energy transfer pathways are opened, which

may compete with the pathways already known from a two-

dye system. The untangling of these pathways not only allows

for a larger FRET range, but also has the potential to study

the proximity relationship of three labeled molecules at the

same time. Given the complexity of protein networks in sig-

naling pathways, such an extension can be quite important in

the quantitative description of protein interactions in signal-

ing processes.

In this article, we present a relatively simple equation

system with which all possible transfer efficiencies can be

calculated in flow cytometry. Previous methods for meas-

uring transfer efficiency between three fluorophores either

relied on complicated fusion protein constructs (27,40–42),

were developed for dyes in solution and not adaptable for

flow cytometers (15,17,23–25,43), or needed a reference

sample to determine the quantity of the donor dye (dye A)

for proper calibration of transfer efficiencies. While the lat-

ter approach is also used and accepted as the simplest

method for calculating transfer efficiency in two-dye systems

(44), it carries the risk of skewed results if the quantity of

the dye changes between samples. In experiments with fluor-

escently labeled antibodies, the probability for this is small

as long as there is no competition between antibodies. How-

ever, when fluorescent protein coupled proteins are

expressed in a cellular system, expression efficiency can vary

from cell-to-cell and this effect is even more accentuated

when multiple exogenous proteins are expressed (45). There-

fore, we sought to develop a method, which does not rely

on an external reference sample to calculate transfer effi-

ciency. We identified and broke down to quantifiable com-

ponents six different emission intensities in total, which, in

a system of equations allow the individual FRET between

each member of the system to be assessed, which in turn

carries information about the relative spatial organization of

the studied molecules or epitopes. Both uncorrected and

competition-corrected transfer efficiencies were calculated to

determine the apparent FRET of the dye system, while still

obtaining the competition-free FRET values of a two-dye

system. In our system, correcting for competition led to

only minimal changes in transfer efficiency. However, in

other systems closer proximity and/or larger spectral overlap

between dyes could result in larger individual FRET efficien-

Figure 3. Three dimensional dot plot and histograms of energy

transfer values determined from a mixture of seven doubly or

triply labeled specimens after a single data acquisition step.

The labeling schemes of the individual specimens can be found in

Table 3.

ORIGINAL ARTICLE

Cytometry Part A � 83A: 375�385, 2013 381

cies and therefore more significant competition, making this

a valuable tool for generating FRET efficiencies comparable

with values from two-dye systems.

In our experiments, we used Alexa Fluor 488, Alexa Fluor

546 and Alexa Fluor 647 fluorophores as dyes A, B, and C,

respectively. There is sufficient spectral overlap between the ex-

Figure 4. Labeling schemes employed to provide alternative spatial distribution and possible transfer routes between fluorescently tagged antibo-

dies. Bold arrows: primary transfer routes, dashed arrows: secondary transfer routes, arrows originating from circles: transfer routes potentially

involved in relay-FRET. [Color figure canbe viewed in the online issuewhich is available atwileyonlinelibrary.com.]

Table 4. Comparison of measured transfer efficiencies (%) calculated with different initial equation sets for labeling schemes described in

Figure 4

AB AC BC RELAY TOTAL

Sample 1

Relay and direct FRET 10.8 (9.9) 9.6 (8.6) 31.4 3.0 11.8

Only relay-FRET 10.8 – 31.5 3.3 12.6

Only direct FRET 7.7 (6.8) 12.6 (11.7) 31.5 – 11.7

No relay or direct FRET 7.7 – 31.5 – 13.3

In two-dye system 10.8 10.2 25.0 – 10.2

Sample 2

Relay and direct FRET 2.7 (2.4) 11.2 (10.9) 43.8 1.0 12.2

Only relay-FRET 2.7 – 43.8 1.1 13.5

Only direct FRET 1.5 (1.3) 12.1 (12.0) 43.8 – 12.0

No relay or direct FRET 1.5 – 43.8 – 13.6

In two-dye system 3.6 10.2 45.1 – 10.2

Sample 3

Relay and direct FRET 22.7 (23.6) –2.8 (–2.2) 22.4 5.0 3.4

Only relay-FRET 22.6 – 22.4 5.0 2.8

Only direct FRET 18.3 (17.8) 3.4 (2.8) 22.4 – 2.8

No relay or direct FRET 18.5 – 22.4 – 3.5

In two-dye system 20.4 2.2 41.0 – 2.2

The displayed EAB and EAC values are noncompetitive FRET values. Competitive FRET values are given in parenthesis where applica-

ble. For standard deviations of transfer efficiencies, please see Section 5 in the Supporting Information.

ORIGINAL ARTICLE

382 TripleFRET

citation and emission spectra of these fluorophores to allow for

all theoretically possible energy transfer routes. When meas-

uring two-dye systems, evaluation with the classical intensity-

based FRET and with the tripleFRET method gave comparable

results. Also, FRET efficiencies obtained by the tripleFRET

approach in three-dye systems for any dye-pair were in good

agreement with the values measured and calculated for the cor-

responding two-dye system. However, when using the two-dye

intensity-based method in a three-dye system, we measured sig-

nificantly lower EAB and significantly higher EAC values com-

pared with the corresponding two-dye systems. This can be

attributed to the quenching of fluorophore intensity and aug-

mentation of sensitized emission by the third fluorophore, so

that distorted values are used as acceptor and donor fluoro-

phore intensities during energy transfer calculation.

To demonstrate the sensitivity and the discrimination

power of our approach, we have mixed, in a single tube, sev-

eral distinctly labeled samples and have shown that following

the acquisition of a single data set it is possible to resolve the

various components of the population based on the correctly

calculated individual FRET efficiencies relevant to the various

molecular interactions characteristic of each label type.

The results also show that while our method is accurate,

it fails to distinguish between different spatial distributions

that produce near-identical transfer efficiency profiles. With-

out prior knowledge of the studied system, based solely on

transfer efficiencies between pertuzumab and trastuzumab in

two-dye systems, Sample 2 in Figure 4 can be assumed to fol-

low the same spatial distribution as Sample 1. Only with the

knowledge of antibody binding stoichiometry (i.e., just one

recognized epitope per protein) can an accurate model be con-

structed. Theoretically, the two cases are distinguished by a

slight increase in EAC and EBC from the presence of additional

transfer routes; however, the contribution of these routes is

mostly small and can be masked by measurement noise and

biological variability.

As with all ensemble-oriented methods relying on signals

from several fluorophores, individual FRET processes are aver-

aged and are indiscernible from one another, that is, a mix of

fluorophore populations displaying large and small FRET can

produce a summed intensity signal indistinguishable from a

homogeneous population with intermediate FRET efficiency,

masking population heterogeneity, resulting in inaccurate

FRET estimations (46). Sample 3 in Figure 4 demonstrates a

spatial distribution where the dominant transfer processes

characterized by EAB and EAC are competitive, and there is an

independent process characterized by EBC. The assumption

that relay transfer is equal to the product of EAB and EBC is still

valid; however, due to spatial separation, one of the processes

contributing to relay-FRET is significantly smaller than the

dominant process characterized by EAB or EBC. In this case,

calculations assuming parallel direct and relay-FRET with the

measured dominant individual transfer values will overesti-

mate quenching of EAB through dye C and contribution of

relay-FRET to sensitized emission of dye C. This in turn

results in underestimation of EAC. If the equation set assumes

only direct transfer from A to C, then EAB is underestimated,

EAC is overestimated and relay-FRET is neglected altogether.

Ideally, the two secondary relay-FRET processes besides the

dominant direct transfers should also be taken into account.

In most cases, various distinct molecular interaction

schemes allow physically plausible EAB, EAC, and EBC values

to be calculated from the same quenched donor and sensi-

tized acceptor emission intensities. This in turn means that

being able to calculate a given transfer efficiency does not

guarantee that the FRET process is actually taking place at

the molecular level. For instance, sensitized emission of dye

C can be attributed to direct FRET between A and C, relay

excitation through B or both. Based solely on intensity data

we cannot distinguish between these cases or tell which one

of them apply to a given situation. Even if multiple orienta-

tions are considered in FRET calculations, as long as the

relative contribution of each to the ensemble FRET signal is

not known, precise efficiency values cannot be calculated.

The same effect is achieved when not all fluorophores parti-

cipate in the transfer process, for instance, when three dif-

ferent proteins are labeled. The presence of single-dye spe-

cies without transfer partners under such conditions is a

problem even in traditional ensemble measurement types

(47). Theoretically, an initial equation set can be developed

to take multiple simultaneous distributions into account;

however, the number of variables does not allow the equa-

tion set to be solved with the six measurable intensities.

Therefore, accurate intensity-based calculations require

prior knowledge about possible transfer routes, either from

measurements in two-dye systems or known and/or limited

spatial distribution of the imaged dyes (for instance rigid

DNA strands that allow for only certain spatial orientations

and limit the number of interacting dyes). Alternatively, sin-

gle-molecule or lifetime measurements can help identify

and characterize possible dye interactions in the studied sys-

tem. Spectral analyses and unmixing may also be a viable

route to determine the relative abundance of different dye

species (42,48). This limitation was not addressed in previ-

ous articles as either the methodology ensured that all three

dyes were within interaction distance with limited possible

relative orientations (all single-molecule imaging methods)

or the chosen system was essentially restricted to certain

relative orientations while ensuring uniform interaction of

dyes (all measurements with DNA strands, fixed distance

three-fluorophore constructs or multimers, where FRET is

only possible in a given relative conformation of the imaged

molecules). In such a fashion, either by chance or design,

the restricted applicability of three-dye FRET measurements

was not unmasked. It should also be noted that these con-

siderations are only vital when precise absolute transfer effi-

ciency values are needed and can be partially neglected

when FRET is only used as a semiquantitative indicator

(e.g., identification of distinct populations, relative confor-

mation changes).

In previous articles, the three-dye system was mostly

characterized with the total energy transfer of the donor

to multiple acceptors. The higher total energy transfer

values of the three-dye system over a two-dye system

ORIGINAL ARTICLE

Cytometry Part A � 83A: 375�385, 2013 383

have been interpreted as an increase in the Forster critical

distance, R0. Using Eqs. (2) and (3), Erelay can be given

as follows:

Erelay ¼1

1þ r6AB

R60AB

� �1þ r6

BC

R60BC

� � ¼ 1

1þ r6ABC

R60ABC

� � ; ð24Þ

where rXY is the actual physical distance of the indicated dye

pair and R0XY is the corresponding Forster distance. The index

ABC denotes the distances as interpreted for the whole relay

transfer process. In a three-dye system, the AC distance as

determined through relay-FRET (which is different from the

Euclidean distance between A and C, since by definition, exci-

tation first has to travel to B before being passed on to C) is

equal to the sum of AB and BC distances:

rABC ¼ rAB þ rBC ð25Þ

By combining Eqs. (24) and (25), the Forster critical distance

for relay transfer can be given as:

R0ABC ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðrAB þ rBCÞ6

6

qR0ABR0BCffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

R60ABr

6BC þ R6

0BCr6AB þ r6ABr

6BC

6p ð26Þ

Therefore, the critical distance for relay-FRET is a function of

the individual specific dye distances. Accordingly, the Forster

distance calculated for relay-FRET is not an intrinsic property of

the dyes determined by their spectra and quantum yields, but an

arbitrary distance derived from distances calculated for two in-

dependent consecutive FRET processes. Thus in our view it is

inappropriate to assign an R0 to relay-FRET, since it is only a

mathematical construct that does not have a true physical mean-

ing, and falsely gives the impression that it possesses the same

type of spatial information as the distances calculated from the

individual two-dye FRETefficiencies in characterizing the three-

dye system. In this sense, relay-FRETshould be used as a qualita-

tive indicator of dye interaction in three-dye systems, but not as

the basis for quantitative distance measurements.

CONCLUSION

The long-standing perception of FRET as a process con-

fined to measuring two fluorophores is gradually being revised

with the development of novel methods for monitoring multi-

ple fluorophores. Most recently, several techniques are emer-

ging for detecting FRET in four dye systems (49–51). However,

even the simpler three-dye methods have not become wide-

spread, chiefly because of unique instrumentation and sample

preparation requirements. In this article, we presented a rela-

tively easy-to-implement method, which can be used with

commercial flow cytometers. Solutions are given for an initial

equation set that allows FRET calculation in a three-dye sys-

tem as is, without the need for an external reference sample to

quantify unquenched donor intensity. We have shown that the

performance of tripleFRET with respect to sample discrimina-

tion and reproduction of FRET efficiency values of two-dye

systems is equivalent to that of three-dye single molecule

FRETmicroscopy methods, and can be applied to FRET inves-

tigations of cell surface proteins labeled with fluorescently

tagged antibodies. Furthermore, limitations arising from

spatial distributions that can influence the interpretation of

experimental data and until now which have not been dis-

cussed in the literature was addressed. This should help

researchers design and conduct FRET experiments to harness

the information gained by the addition of a third dye to con-

ventional two-dye systems.

LITERATURE CITED

1. Forster T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys(Leipzig) 1948;2:55–75.

2. Stryer L, Haugland RP. Energy transfer: A spectroscopic ruler. Proc Natl Acad SciUSA 1967;58:719–726.

3. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem1978;47:819–846.

4. Nagy P, Bene L, Balazs M, Hyun WC, Lockett SJ, Chiang NY, Waldman F, FeuersteinBG, Damjanovich S, Szollosi J. EGF-induced redistribution of erbB2 on breast tumorcells: Flow and image cytometric energy transfer measurements. Cytometry1998;32:120–131.

5. Sorkin A, McClure M, Huang F, Carter R. Interaction of EGF receptor and grb2 inliving cells visualized by fluorescence resonance energy transfer (FRET) microscopy.Curr Biol 2000;10:1395–1398.

6. Leray A, Spriet C, Trinel D, Heliot L. Three-dimensional polar representation formultispectral fluorescence lifetime imaging microscopy. Cytometry Part A2009;75A:1007–1014.

7. Spriet C, Trinel D, Riquet F, Vandenbunder B, Usson Y, Heliot L. Enhanced FRETcontrast in lifetime imaging. Cytometry Part A 2008;73A:745–753.

8. Vamosi G, Damjanovich S, Szollosi J. Dissecting interacting molecular populationsby FRET. Cytometry Part A 2008;73A:681–684.

9. Berney C, Danuser G. FRET or No FRET: A Quantitative Comparison. Biophys J2003;84:3992–4010.

10. Szollo†si J, Nagy P, Sebestyen Z, Damjanovich S, Park JW, Matyus L. Applications offluorescence resonance energy transfer for mapping biological membranes. J Biotech-nol 2002;82:251–266.

11. Jares-Erijman EA, Jovin TM. FRET imaging. Nat Biotechnol 2003;21:1387–1395.

12. Kim J, Li X, Kang MS, Im KB, Genovesio A, Grailhe R. Quantification of proteininteraction in living cells by two-photon spectral imaging with fluorescent proteinfluorescence resonance energy transfer pair devoid of acceptor bleed-through. Cyto-metry Part A 2012;81A:112–119.

13. Paar C, Paster W, Stockinger H, Schutz GJ, Sonnleitner M, Sonnleitner A. Highthroughput FRET screening of the plasma membrane based on TIRFM. CytometryPart A 2008;73A:442–450.

14. Liu J, Lu Y. FRET study of a trifluorophore-labeled DNAzyme. J Am Chem Soc2002;124:15208–15216.

15. Ohya Y, Yabuki K, Hashimoto M, Nakajima A, Ouchi T. Multistep fluorescence reso-nance energy transfer in sequential chromophore array constructed on oligo-DNAassemblies. Bioconjug Chem 2003;14:1057–1066.

16. Watrob HM, Pan CP, Barkley MD. Two-step FRET as a structural tool. J Am ChemSoc 2003;125:7336–7343.

17. Aneja A, Mathur N, Bhatnagar PK, Mathur PC. Triple-FRET technique for energytransfer between conjugated polymer and TAMRA dye with possible applications inmedical diagnostics. J Biol Phys 2008;34:487–493.

18. Clamme JP, Deniz AA. Three-color single-molecule fluorescence resonance energytransfer. Chemphyschem 2005;6:74–77.

19. Ernst S, Duser MG, Zarrabi N, Borsch M. Three-color Forster resonance energytransfer within single F(0)F(1)-ATP synthases: Monitoring elastic deformations ofthe rotary double motor in real time. J Biomed Opt 2012;17:011004.

20. Galperin E, Verkhusha VV, Sorkin A. Three-chromophore FRET microscopy to ana-lyze multiprotein interactions in living cells. Nat Methods 2004;1:209–217.

21. Haustein E, Jahnz M, Schwille P. Triple FRET: A tool for studying long-range molec-ular interactions. Chemphyschem 2003;4:745–748.

22. Hohng S, Joo C, Ha T. Single-molecule three-color FRET. Biophys J 2004;87:1328–1337.

23. Lee NK, Kapanidis AN, Koh HR, Korlann Y, Ho SO, Kim Y, Gassman N, Kim SK,Weiss S. Three-color alternating-laser excitation of single molecules: Monitoringmultiple interactions and distances. Biophys J 2007;92:303–312.

24. Navarathne D, Ner Y, Grote JG, Sotzing GA. Three dye energy transfer cascade withinDNA thin films. Chem Commun (Camb) 2011;47:12125–12127.

25. Tong AK, Jockusch S, Li Z, Zhu HR, Akins DL, Turro NJ, Ju J. Triple fluorescenceenergy transfer in covalently trichromophore-labeled DNA. J Am Chem Soc2001;123:12923–12924.

26. Galperin E, Sorkin A. Visualization of Rab5 activity in living cells using FRETmicros-copy. Methods Enzymol 2005;403:119–134.

27. He L, Wu X, Simone J, Hewgill D, Lipsky PE. Determination of tumor necrosis factorreceptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRETdetected by flow cytometry. Nucleic Acids Res 2005;33:e61.

ORIGINAL ARTICLE

384 TripleFRET

28. Lee S, Lee J, Hohng S. Single-molecule three-color FRETwith both negligible spectraloverlap and long observation time. PLoS One 2010;5:e12270.

29. Shyu YJ, Suarez CD, Hu CD. Visualization of AP-1 NF-kappaB ternary complexes inliving cells by using a BiFC-based FRET. Proc Natl Acad Sci USA 2008;105:151–156.

30. Fazekas Z, Petras M, Fabian A, Palyi-Krekk Z, Nagy P, Damjanovich S, Vereb G, Szol-losi J. Two-sided fluorescence resonance energy transfer for assessing molecular inter-actions of up to three distinct species in confocal microscopy. Cytometry Part A2008;73A:209–219.

31. Mocanu MM, Fazekas Z, Petras M, Nagy P, Sebestyen Z, Isola J, Timar J, Park JW,Vereb G, Szollosi J. Associations of ErbB2, beta1-integrin and lipid rafts on Herceptin(Trastuzumab) resistant and sensitive tumor cell lines. Cancer Lett 2005;227:201–212.

32. Shrestha D, Bagosi A, Szollosi J, Jenei A. Comparative study of the three differentfluorophore antibody conjugation strategies. Anal Bioanal Chem 2012;404:1449–1463.

33. Szentesi G, Horvath G, Bori I, Vamosi G, Szollosi J, Gaspar R, Damjanovich S, JeneiA, Matyus L. Computer program for determining fluorescence resonance energytransfer efficiency from flow cytometric data on a cell-by-cell basis. Comput MethodsPrograms Biomed 2004;75:201–211.

34. Nagy P, Vamosi G, Bodnar A, Lockett SJ, Szollosi J. Intensity-based energy transfermeasurements in digital imaging microscopy. Eur Biophys J 1998;27:377–389.

35. Roszik J, Lisboa D, Szollosi J, Vereb G. Evaluation of intensity-based ratiometricFRET in image cytometry-approaches and a software solution. Cytometry Part A2009;75A:761–767.

36. Nagy P, Bene L, Hyun WC, Vereb G, Braun M, Antz C, Paysan J, Damjanovich S,Park JW, Szollsi J. Novel calibration method for flow cytometric fluorescence reso-nance energy transfer measurements between visible fluorescent proteins. CytometryPart A 2005;67A:86–96.

37. Vamosi G, Baudendistel N, von der Lieth CW, Szaloki N, Mocsar G, Muller G, BrazdaP, Waldeck W, Damjanovich S, Langowski J, et al. Conformation of the c-Fos/c-Juncomplex in vivo: A combined FRET, FCCS, and MD-modeling study. Biophys J2008;94:2859–2868.

38. Tron L, Szollosi J, Damjanovich S, Helliwell SH, Arndt-Jovin DJ, Jovin TM. Flowcytometric measurement of fluorescence resonance energy transfer on cell surfaces.Quantitative evaluation of the transfer efficiency on a cell-by-cell basis. Biophys J1984;45:939–946.

39. Bagossi P, Horvath G, Vereb G, Szollosi J, Tozser J. Molecular modeling of nearly full-length ErbB2 receptor. Biophys J 2005;88:1354–1363.

40. Lopez-Gimenez JF, Canals M, Pediani JD, Milligan G. The alpha1b-adrenoceptorexists as a higher-order oligomer: Effective oligomerization is required for receptormaturation, surface delivery, and function. Mol Pharmacol 2007;71:1015–1029.

41. Milligan G, Pediani JD, Canals M, Lopez-Gimenez JF. Oligomeric structure of thealpha1b-adrenoceptor: Comparisons with rhodopsin. Vision Res 2006;46:4434–4441.

42. Sun Y, Wallrabe H, Booker CF, Day RN, Periasamy A. Three-color spectral FRETmi-croscopy localizes three interacting proteins in living cells. Biophys J 2010;99:1274–1283.

43. Klostermeier D, Sears P, Wong CH, Millar DP, Williamson JR. A three-fluorophoreFRET assay for high-throughput screening of small-molecule inhibitors of ribosomeassembly. Nucleic Acids Res 2004;32:2707–2715.

44. He L, Olson DP, Wu X, Karpova TS, McNally JG, Lipsky PE. A flow cytometricmethod to detect protein-protein interaction in living cells by directly visualizing do-nor fluorophore quenching during CFP->YFP fluorescence resonance energy transfer(FRET). Cytometry Part A 2003;55A:71–85.

45. Goedhart J, van Weeren L, Adjobo-Hermans MJ, Elzenaar I, Hink MA, Gadella TWJr. Quantitative co-expression of proteins at the single cell level-application to a mul-timeric FRET sensor. PLoS One 2011;6:e27321.

46. Dietrich A, Buschmann V, Muller C, Sauer M. Fluorescence resonance energy transfer(FRET) and competing processes in donor-acceptor substituted DNA strands: Acomparative study of ensemble and single-molecule data. J Biotechnol 2002;82:211–231.

47. Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR, Weiss S. Probing the interactionbetween two single molecules: Fluorescence resonance energy transfer between a sin-gle donor and a single acceptor. Proc Natl Acad Sci USA 1996;93:6264–6268.

48. Ecker RC, de Martin R, Steiner GE, Schmid JA. Application of spectral imaging mi-croscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.Cytometry Part A 2004;59A:172–181.

49. Kim SH, Gunther JR, Katzenellenbogen JA. Monitoring a coordinated exchange pro-cess in a four-component biological interaction system: Development of a time-resolved terbium-based one-donor/three-acceptor multicolor FRET system. J AmChem Soc 2010;132:4685–4692.

50. Lee J, Lee S, Ragunathan K, Joo C, Ha T, Hohng S. Single-molecule four-color FRET.Angew Chem Int Ed Engl 2010;49:9922–9925.

51. Stein IH, Steinhauer C, Tinnefeld P. Single-molecule four-color FRET visualizesenergy-transfer paths on DNA origami. J Am Chem Soc 2011;133:4193–4195.

ORIGINAL ARTICLE

Cytometry Part A � 83A: 375�385, 2013 385