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q 2005 Wiley-Liss, Inc. Cytometry Part A 65A:148–157 (2005)

Selecting the Right Fluorophores and Flow

Cytometer for Fluorescence Resonance Energy
Transfer Measurements
G
ath,1 Mikl

abor Horv
os Petr


as,1 Gergely Szentesi,2 Akos F
an,1 John W. Park,3
abi

Gy€ 1
orgy Vereb, and J
anos Sz€ osi *
oll} 1,2
1
Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
2
Cell Biophysical Research Group of Hungarian Academy of Sciences, Research Center for Molecular Medicine,
Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
3
Division of Hematology/Oncology, University of California, San Francisco, San Francisco, California
Received 21 December 2004; Revision Received 8 February 2005; Accepted 10 February 2005

Background: Fluorescence resonance energy transfer
applied in flow cytometry (FCET) is an excellent tool for
determining supramolecular organization of biomolecules
at the cell surface or inside the cell. Availability of new
fluorophores and cytometers requires the establishment
of fluorophore dye pairs most suitable for FCET measure-
ments.
Methods: A gastric tumor cell line (N87) was labeled for
major histocompatibility complex class I heavy chain and
b2-microglobulin with antibodies conjugated with fluores-
cein- and indocarbocyanine-like fluorophores and analyzed
in FCET measurements on a cell-by-cell basis using three
flow cytometers: FACSCalibur, FACSDiVa, and FACSArray.
Results: Normalized fluorescence intensity values were
measured and normalized energy transfer efficiencies,
spectral overlap integrals, and crucial dye- and instrument-
dependent parameters were calculated for all matching
pairs of seven fluorophores on the three commercial cyt-
ometers. The most crucial parameter in determining the
applicability of the donor-acceptor pairs was the normal-
ized fluorescence intensity and the least important one
was the spectral overlap.
Conclusions: On the basis of available laser lines, the
optimal dye pair for all three cytometers is the Alexa546-
Alexa647 pair, which produces high energy transfer effi-
ciency values and has the best spectral characteristics
with regard to laser excitation, detection of emission, and
spectral overlap. q 2005 Wiley-Liss, Inc.
Key terms: fluorescence resonance energy transfer; flow
cytometry; donor-acceptor pair

Investigation of protein-protein associations is impor-
tant in understanding the structure-function relations in
living cells. Fluorescence resonance energy transfer
(FRET) based methods are excellent tools for determining
association patterns of biomolecules at the cell surface or
inside the cell. With the help of FRET, molecular dimen-
sions can be measured and determined in functioning, live
cells, thus providing information that would be impossible
to obtain with other classic approaches, e.g., electron
microscopic methods.
The theory of FRET was introduced by F€ orster in the
late 1940s (1); however, it did not become widespread in
biomedical studies until Stryer’s popularizing article (2).
The FRET process is a long-range dipole-dipole interaction
in which an excited donor fluorophore transfers its energy
to a neighboring acceptor molecule in a nonradiative way.
The main application of FRET as a spectroscopic ruler is
based on the fact that the rate of energy transfer depends
on the inverse sixth power of the separation of the two
interacting molecules. The most apparent manifestation
of the FRET process is the decrease of donor fluorescence
in the presence of an acceptor in close vicinity (1–10 nm),
which is accompanied by an increase of fluorescence of
the acceptor (if the acceptor is a fluorescent molecule).
The FRET phenomenon has been adapted for microscopy
(3–6) and flow cytometry (7–9) and several biological
Contract grant sponsor: Fogarty International Collaboration Award;
Contract grant number: 1 RO2 TW00871-01A2; Contract grant sponsor:
European Community; Contract grant number: EU FP6 LSHB-CT-2004-
503467; Contract grant sponsor: Hungarian Ministry of Health; Contract
grant numbers: ETT 524/2003 and ETT 532/2003; Contract grant spon-
sor: Hungarian National Research Fund; Contract grant numbers: OTKA
T030399 and T037831; Contract grant sponsor: Hungarian Ministry of
Education; Contract grant number: B
ek
esy Fellowship to G.V.
*Correspondence to: J
anos Sz€ oll}
osi, Department of Biophysics and
Cell Biology, Faculty of Medicine, Medical and Health Science Center, Uni-
versity of Debrecen, P.O. Box 39, Nagyerdei krt. 98, H-4012 Debrecen,
Hungary.
E-mail: szollo@jaguar.dote.hu
Published online 11 April 2005 in Wiley InterScience (www.interscience.
wiley.com).
DOI: 10.1002/cyto.a.20142

FLUOROPHORES FOR FLOW CYTOMETRIC FRET
structures have been successfully investigated: receptors
involved in immune response (10–12), growth factor
receptors on tumor cells (13–15), determination of pro-
tein conformation (16,17), and lipid microdomain struc-
tures (9,18,19). Several reviews are also available for bio-
medical and clinical applications of FRET (8,18,20–24).
One of the first FRET approaches carried out in flow
cytometry was the flow cytometric energy transfer (FCET)
method with two-color excitation and proper handling of
spectral crosstalk between fluorescent dyes (7,25). This
method was developed for a classic cytometer equipped
with 488- and 514-nm lasers suitable for the frequently
applied fluorescein and rhodamine dye pair. During FCET
measurements, these excitation wavelengths are not opti-
mal for the fluorescein and rhodamine pair because of the
substantial crosstalk between the dyes in various detec-
tion channels. In new versions of flow cytometric sys-
tems, which usually incorporate 488- and 635-nm laser
excitations, the fluorescein and rhodamine pair cannot be
applied because the 635-nm excitation is outside the spec-
tral range of rhodamine and the spectral overlap between
fluorescein and a typical dye excitable at 635 nm is mini-
mal, practically prohibiting the FRET process. However,
recently developed dye pairs (Cy3-Cy5) were successfully
adopted to a newer, bench-top cytometer (FACSCalibur),
and the analysis procedures were improved to consider
autofluorescence on a cell-by-cell basis by means of an
otherwise unused detection channel (26).
In recent years, the availability of cheaper diode and
solid-state lasers with higher power output and matching
fluorophore pairs, which span the entire visible spectrum
reaching the far-red region, raised the question of select-
ing the ideal dye pair for the instrument in FRET studies.
In our study, we investigated the performance of fluores-
cent dyes: Cy3, Alexa546, Alexa555, and Alexa568 (FRET
donors) and Cy5, Alexa633, and Alexa647 (FRET accep-
tors) in studying molecular associations with the help
of various flow cytometers: FACSCalibur, FACSDiVa, and
FACSArray. Some other types of fluorescent molecules
were not included in this investigation, but their general
roles and usefulness were also considered. In this study,
we have summarized the results obtained for all matching
pairs of the seven fluorophores on three flow cytometers
and established parameters crucial for FCET measure-
ments. Taking into consideration all the significant para-
meters that influence the magnitude and accuracy of the
FRET efficiency value measured, we determined the most
suitable donor-acceptor pair for FCET measurements in all
cytometers tested.
MATERIALS AND METHODS
Cell Lines
Human gastric cancer cell line N87 with high major his-
tocompatibility complex (MHC) class I expression level
was obtained from the American Type Culture Collection
(Rockville, MD, USA) and grown according to the manu-
facturer’s specification (in RPMI containing 10% fetal calf
serum, 2 mM l-glutamine, and 0.25% gentamicin in 5%
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149
CO2 atmosphere) to confluency. For flow cytometry, cells
were harvested by treatment with 0.05% trypsin and
0.02% ethylenediaminetetraacetic acid.
Conjugation of Antibodies With Fluorescent Dyes
The W6/32 (against MHC class I heavy chain) and L368
(against b2-microglobulin) antibodies were a kind gift
from Francis Brodsky. Aliquots of antibodies (
1 mg/ml
concentration) were conjugated with the succinimide
derivative of fluorescein-like (Alexa546, Alexa555,
Alexa568, Alexa633, and Alexa647; Molecular Probes,
Eugene, OR, USA) and of indocarbocyanine-like (Cy3 and
Cy5; Amersham Biosciences, Freiburg, Germany) dyes, as
described previously (27). In the experiments, the W6/32
antibody was labeled with donor fluorophores (Cy3,
Alexa546, Alexa555, and Alexa568), and the L368 anti-
body with acceptor fluorophores (Cy5, Alexa633, and
Alexa647). The labeling ratios (L) were in the range of 1 to
5, where concentration quenching does not yet have a sig-
nificant effect on the fluorescence quantum yield of the
donor and acceptor dyes.
Labeling Cells with Fluorescent Antibodies
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 final concentration of 4 3 107
cells/ml, and then 25 ml of conjugated antibodies was
added to 25 ml of cell suspension and cells were incubated
for 30 min on ice. The excess of antibody was at least five-
fold above saturating concentrations during the incuba-
tion. The same procedure was used for FRET samples, in
which a mixture of donor- and acceptor-labeled antibodies
was added to the cell suspension. The labeled cells were
washed twice with excess cold PBS and fixed with 1% for-
maldehyde and PBS.
Flow Cytometers
The FCET measurements were carried out on three
commercial cytometers: FACSCalibur, FACSVantage SE
with DiVa option, and FACSArray (Becton Dickinson,
Franklin Lakes, NJ, USA). The FACSCalibur is equipped
with a 488- and a 635-nm laser and three detectors were
used: FL2 (585/42 band pass), FL3 (670 long pass), and
FL4 (661/16 band pass filter). The FACSDiVa features a
532-nm diode-pumped solid-state laser, a 633-nm air-
cooled HeNe laser, and a 488-nm Ar laser for forward scat-
ter triggering. The emission filters were a 585/42 band
pass filter and two 650 long pass filters. The FACSArray is
shipped with a 532-nm solid-state laser and a 635-nm
diode laser, and for FRET measurements the detectors
with 585/42 band pass (yellow), 685 long pass (far red),
and 661/16 band pass (red) filters were used.
Theory of Fluorescence Resonance Energy Transfer
The FRET phenomenon is a dipole-dipole interaction in
which an excited donor fluorophore transfers its energy
to a neighboring acceptor molecule in a nonradiative way
 15524930, 2005, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20142 by Centro De Investigacion Y De Estudios Avanzados Del Instituto, Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
150

HORVATH ET AL.

(28). This interaction manifests in the decrease of lifetime

of the donor molecule. The measure of FRET is the effi-
ciency (E) of energy transfer:

kT
E¼ ð1Þ
kT þ kF þ kn

i.e., the fraction of absorbed photons that are transferred

without radiation to the acceptor, where kT is the rate of
energy transfer, kF is the rate of fluorescence of the donor
without an acceptor, and kn is the sum of all nonradiative
decay rates of the donor except of energy transfer. The
rate of interaction can be described as follows:

6
R0
kT ¼ ðkF þ kn Þ ð2Þ
R

The combination of these two equations provides the

distance dependence of FRET efficiency:

R06
E¼ ð3Þ
R06 þ R6

where R0 is the so-called F€

orster distance, i.e., the distance
at which FRET efficiency is 50%. The following term was FIG. 1. Normalized excitation spectra of donor and acceptor fluoro-
phores. The three laser lines used in our flow cytometric setups are indi-
derived for R0 (nanometers): cated by vertical lines as wavelength of excitation.

R0 ¼ 978  ðQD k2 n4 JDA Þ1=6 ; ð4Þ Flow Cytometric Energy Transfer Measurements
The easiest way to measure FRET in a flow cytometer is
where QD is the donor quantum yield in the absence of to use the background corrected fluorescence signal of
acceptor dyes, n is the index of refraction of the convey- the donor from a donor-only labeled sample (IDD) and a
ing medium (usually 1.4 is used for cell surface antibody donor- and acceptor-labeled sample (IDDA) and to calcu-
labeling), k2 is the orientation factor (its value is 0 to 4 late FRET efficiency (E) as:
and equals 2=3 for dynamic averaging of isotropic transition
moments) (29,30), and JDA is the overlap integral (28). IDDA
The JDA overlap integral (moles per cubic centimeter) can E ¼1 ð7Þ
IDD
be calculated in the knowledge of the donor emission and
acceptor excitation spectra:
The disadvantage of this ‘‘donor quenching’’ method is
Z 1 that the mean fluorescence intensity of two separate sam-
JDA ¼ fD ðlÞ«A ðlÞl4 dl; ð5Þ ples is used and these intensities are not corrected for
0 spectral crosstalk from the acceptor, and corrections for
antibody competition also have to be considered.
where fD(l) is the normalized fluorescence spectrum of To make corrections for spectral crosstalk and be able
the donor dye: to measure FRET efficiency on a cell-by-cell basis, one has
to detect three independent signals (7):
FDl ðlÞ
fD ðlÞ ¼ R 1 ð6Þ S4
0 FDl ðlÞdl I1 ðexcD ; emD Þ ¼ ID ð1  EÞ þ IA S4 þ  ID Ea ð8Þ
S2

The overlap integrals of the dye pairs applied in this I2 ðexcD ; emA Þ ¼ ID ð1  EÞS1 þ IA S2 þ ID Ea ð9Þ
study were calculated from spectroscopic data available at
the Web site of the manufacturer (http://www.probes. S3
I3 ðexcA ; emA Þ ¼ ID ð1  EÞS3 þ IA þ  ID Ea ð10Þ
com), except for Cy3 and Cy5, which were measured on a S1
FluoroLog-3 spectrofluorometer (Jobin Yvon, Edison, NJ,
USA). The excitation spectra of the fluorophores can be The equation set contains the direct donor excitation in I1,
seen in Figure 1. the acceptor sensitization in I2, the direct acceptor excita-
 15524930, 2005, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20142 by Centro De Investigacion Y De Estudios Avanzados Del Instituto, Wiley Online Library on [26/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
FLUOROPHORES FOR FLOW CYTOMETRIC FRET 151
Table 1
Normalized Fluorescence Intensities and Molar Extinction Coefficients of Donor and Acceptor
Fluorophores*
Fluorophore Imeas L Inorm emax (1/M* cm) eexc (1/M* cm)
FACSCalibur
Donor (488 nm) — 8a
Cy3 124 3.56 33 150,000 28,000
Alexa546 100 1.85 50 112,000 8,000
Alexa555 147 3.46 40 158,000 23,000
Alexa568 29 4.19 5 88,000 5,000
Acceptor (635 nm) — 1a
Cy5 411 2.91 158 250,000 187,000
Alexa633 80 1.06 75 159,000 154,000
Alexa647 1054 5.2 202 250,000 141,000
FACSDiVa
Donor (532 nm) — 27a
Cy3 1737 3.56 481 150,000 86,000
Alexa546 2094 1.85 1118 112,000 37,000
Alexa555 2215 3.46 632 158,000 78,000
Alexa568 280 4.19 61 88,000 30,000
Acceptor (633 nm) — 8a
Cy5 1130 2.91 386 250,000 174,000
Alexa633 392 1.06 362 159,000 159,000
Alexa647 2954 5.2 567 250,000 129,000
*Imeas, measured fluorescence intensity (in arbitrary units); L, labeling ratio of antibody (W6/32 in
the case of donor and L368 in the case of acceptor labeling); Inorm, normalized fluorescence inten-
sity; emax, molar extinction coefficient at maximum intensity; eexc, molar extinction coefficient at
excitation wavelength.
a
Autofluorescence.

tion in I3, the unquenched donor fluorescence (ID), and the
pure acceptor signal (IA). All intensities are background cor-
rected. To solve the equation set, one has to determine the
S1–4 spectral overlap factors in separate measurements with
donor- and acceptor-only labeled samples. Thereby the fol-
lowing equation can be derived for FRET efficiency:
E
A¼
1E
1 S1 S2 ½I2 ð1  S3 S4 Þ  I1 ðS1  S2 S3 Þ  I3 ðS2  S1 S4 Þ
¼ 
a ðS1  S2 S3 ÞðI1 S2  I2 S4 Þ
ð11Þ
The a-factor is determined empirically according to the
following formula:
I2A LD BD «D
a¼  ð12Þ
I1D LA BA «A
where I2A is the I2 signal of acceptor-only labeled sample and
I1D is the I1 signal of donor-only labeled sample, L is the label-
ing ratio of the antibodies, B is the mean number of recep-
tors labeled, and e is the excitation coefficient of the dyes at
the donor excitation wavelength (the D and A indexes refer
to donor and acceptor, respectively). The ratio of molar
extinction coefficients of the fluorophores at the donor exci-
tation wavelength (eD/eA) is the so-called efr coefficient.
The E transfer efficiency values are calculated on a cell-
by-cell basis with AFlex software (31) and are presented
as mean values from approximately normally distributed,
unimodal energy transfer histograms of 20,000 cells.
RESULTS
Establishing Factors That Influence
FCET Measurements
We found four parameters that are relevant for deter-
mining the practicality of FRET dye pairs, namely normal-
ized fluorescence intensity (Inorm), overlap integral (JDA),
normalized energy transfer efficiency (Enorm), and the efr
coefficient.
The fluorescence intensity is very important in FCET mea-
surements because one measure of FRET efficiency is the
:
decrease of donor signal in the presence of an acceptor.
Thus, it is preferable that the donor signals are at least twice
as high as the background intensities to detect donor
quenching. In these measurements, the fluorescence intensi-
ties were at least three times, but mostly fifty times, above
background intensity. The background subtracted intensities
were corrected for labeling ratios to obtain the fluorescence
intensities for the same number of fluorescent dyes (Table
1). The Inorm values were in the ranges of 5 to 200 and 60 to
1,100 for FACSCalibur and FACSDiVa, respectively.
Because the rate of energy transfer is influenced by the
F€orster distance of the dye pair, which contains physical
constants distinctive for the dyes (see equation 4), this
parameter should be a good tool for characterizing the
FRET dye pairs. Unfortunately, the quantum yield highly
depends on the environment, so it is difficult to tell the
value of QD for a given donor dye under measurement
conditions. However, it should be noted that R0 is mostly
sensitive to QD in the range of 0.0 to 0.3 (28), which is the
characteristic range for cyanine dyes, whereas the Alexa
dyes have quantum yield values about or higher than 0.3
in our experience (data not shown); for further informa-
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152

HORVATH ET AL.

Table 2 portional to the labeling ratios (or the a-factor) but the A
Overlap Integrals of Donor and Acceptor Dye Pairs* value is, the A 5 E/(1 2 E) values have to be used for nor-
Acceptors malization to LD:LA 5 1:1. The normalized efficiencies
Donors Cy5 Alexa633 Alexa647
(Enorm) listed in Table 3 were calculated with the formulas
derived from equations 11 and 12:
Cy3 5.5 5.5 5.2
Alexa546 8.1 7.2 7.8
LR E
Alexa555 8.7 7.4 8.6 Anorm ¼  ð13Þ
Alexa568 14.6 10.5 14.9 LRnorm 1  E
*Overlap integrals (JDA; 1013 M/cm3) are calculated from emis-
sion and excitation spectra with equation 5 as explained in Mate- where LRnorm 5 1. Then, the obtained Anorm values were
rials and Methods. converted to normalized transfer efficiencies:

tion, please see Shapiro (32), pp. 336–338. In the present
study, the actual distances were not important, so we used
the overlap integral rather than the F€orster distance. The
JDA overlap integrals for the 12 combinations were calcu-
lated as described in Materials and Methods and are pre-
sented in Table 2. The JDA values were in the range of
5–15 3 10213 M/cm3 and were the highest for dye pairs
containing Alexa568 as a donor and lowest for pairs con-
taining Cy3 as a donor.
The actual measured FRET efficiency (E) also has to be
taken into account to find a good dynamic range for the E
value. Low E values compromise the statistical accuracy
of the measurements and hinder monitoring changes in
the association pattern. High E values are also error prone,
especially above 90%, because of the way E is calculated
from the experimentally determined A parameter (E 5 A/
[1 1 A]). The E values were obtained by detecting all
three intensities in equations 8–10, solving for equation
11 on a cell-by-cell basis, and tabulating the means of
unimodal efficiency distribution histograms. To compare
the efficiency values between dye pairs, they have to be
normalized for their excitation spectrum, quantum yield,
and the donor-acceptor ratios of the dyes. The excitation
spectra are already considered in the a-factor, although
the quantum yields are not known, but the correction for
donor-acceptor ratios can be easily implemented. In our
biological system, the light and heavy chains of MHC class
I molecules are expressed constitutively on the cell sur-
face, and only a small portion (<10%) of the heavy chains
stands alone. So it can be surmised that almost all donor-
labeled antibodies are in close proximity of 1 and only one
acceptor-labeled antibody. In such cases, only the ratio of
the labeling ratios (LD/LA 5 LR) has to be considered
because the ratio of the number of antibodies is approxi-
mately 1. Because the FRET efficiency is not linearly pro-
Anorm
Enorm ¼ ð14Þ
1 þ Anorm
Dye pairs containing Alexa568 as a donor showed the
highest FRET efficiency values; on one occasion, for the
Alexa568 and Alexa633 pair, the normalized FRET effi-
ciency value was even close to 0.80 on the FACSDiVa
instrument. The lowest normalized FRET efficiency values
(about 0.22) were obtained for dye pairs containing
Alexa546 dyes as a donor on the FACSCalibur instrument.
FRET efficiency histograms for the dye pairs containing
Alexa647 as an acceptor are shown in Figure 2. The high-
est mean value of the FRET efficiency distribution histo-
gram was obtained for the Alexa568-Alexa647 pair, but
the width of the distribution curve was also the largest for
this pair.
Another important factor is the a-factor or the efr coeffi-
cient. Because the a-factor contains parameters that may
vary in each experiment, we found that the efr coefficient
provides a more reliable means to characterize FRET dye
pairs. The efr coefficient is given by the ratio of molar
extinction coefficients of the fluorophores at the donor
excitation wavelength (eD/eA). Because this physical para-
meter cannot be reliably obtained from observing the
absorption spectrum of the dyes, another possibility is to
determine the FRET efficiency from equation 11 and
change the efr factor (or the related a-factor) until the effi-
ciency value equals the one obtained by the donor
quenching calculation (corrected for antibody competi-
tion) and/or the unquenched donor fluorescence intensity
(ID) equals the fluorescence intensity of the donor-only
labeled sample. The efr coefficients determined this way
are summarized in Table 4 for two cytometers (FACSCali-
bur and FACSDiVa). The efr values were in the range of
0.2 to 17 and were the highest for dye pairs containing

Table 3
Normalized Energy Transfer Efficiencies (Enorm) of Dye Pairs*
Acceptors
FACSCalibur FACSDiVa
Donors Cy5 Alexa633 Alexa647 Cy5 Alexa633 Alexa647
Cy3 0.341 0.327 0.298 0.345 0.326 0.244
Alexa546 0.236 0.213 0.202 0.311 0.412 0.258
Alexa555 0.366 0.382 0.393 0.388 0.537 0.451
Alexa568 0.574 0.641 0.585 0.655 0.796 0.657
*The E values were normalized to labeling ratios of donor and acceptor dyes.

FLUOROPHORES FOR FLOW CYTOMETRIC FRET
FIG. 2. FRET efficiency distribution histograms of donor fluorophores
with Alexa647 as an acceptor measured on a FACSDiVa flow cytometer.
The FRET efficiency values were calculated on a cell-by-cell basis by equa-
tion 11 as described in Material and Methods.
Cy3 as a donor and lowest for pairs containing Alexa568
as a donor.
Selecting the Most Suitable FRET Dye Pair
Because of the large number of combinations between
the four donor and three acceptor dyes, we wanted to nar-
row the scope of our search to obtain the most suitable
dye pair. The relevant approach seemed to be to focus first
on the overlap integrals and FRET efficiency values to
sieve out the less favorable dyes. As can be seen in Table
2, Cy3 has the worst JDA characteristics and is known to
have the lowest fluorescence quantum yield (see product
description), although the Enorm and efr values are quite
high and the intensity is average. Another striking feature
is the very high JDA and E values for the Alexa568 dye.
However, these values should be contrasted with the
extremely low intensity and efr values, which result in less
reliable FRET efficiency histograms (broad distribution)
and render FCET measurements practically useless with
this dye. For a comparison of FRET efficiency distribution
histograms of donor dyes with the acceptor Alexa647, see
Figure 2. Thus, two donor dyes, Cy3 and Alexa568, should
be excluded from further investigations. Looking at the
data of acceptor dyes, we found no significant differences.
Although Alexa647 had slightly smaller Enorm values, it
produced the highest intensity, whereas Alexa633 had
Table 4
The efr Coefficients of FRET Dye Pairs*
FACSCalibur
Donors Cy5 Alexa633 Alexa647
Cy3 17.0 10.4 14.8
Alexa546 9.0 7.1 8.1
Alexa555 12.7 6.8 9.2
Alexa568 1.1 0.7 0.9
*The efr coefficient is the ratio of molar extinction coefficients (eD/eA) of the fluorophores at
the donor excitation wavelength (488 nm on FACSCalibur and 532 nm on FACSDiVa). The values
were determined empirically by comparing the FRET efficiency values obtained on a cell-by-cell
basis with that obtained by the donor quenching method.
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153
lower JDA and efr values, but its Enorm was remarkable. So
none of the acceptors should be excluded.
For a more thorough investigation, Alexa546 and
Alexa555, as donors, and Cy5, Alexa633, and Alexa647, as
acceptors, giving six dye pairs, were used.
First, we analyzed the intensities (Table 1) and found
that Alexa546 had a fluorescence intensity that was two
times as high as that for Alexa555. Among the acceptors,
we found no great differences and the intensities fluctu-
ated a bit from one cytometer to another. Nonetheless, it
should be noted that Alexa647 has the highest intensity
and Alexa633 the lowest intensity. So the Alexa546-
Alexa647 pair is the best choice when considering fluores-
cence intensity.
Second, we examined the overlap integrals (Table 2)
because they determine the range of sensitivity of energy
transfer via the F€
orster distance. In all cases, Alexa633 as
an acceptor produced the smallest overlap integral and
Alexa555 as a donor had the highest overlap integral. The
other two acceptors, Cy5 and Alexa647, produced similar
values, although the value for Cy5 was a bit higher. Thus,
in this respect, the Alexa555-Cy5 pair was the best dye
pair, but the other dyes should not be excluded because
of the small differences between dye pairs (JDA values
were in the range of 7.2 to 8.7 3 10213 M/cm3).
Then we investigated the normalized FRET efficiency
values (Table 3). For the two donor dyes, we obtained
fairly similar values on both cytometers, so we do not
believe this is a decisive factor in either case. However,
there were larger differences between the acceptor dyes,
and Alexa633 had the highest FRET values, whereas
Alexa647 had the lowest. Thus the Alexa555-Alexa633
pair is the most effective when viewing the normalized
FRET efficiency values. It should be noted that the Enorm
values measured on FACSDiVa (532- and 633-nm excita-
tion) were higher in all cases than those measured on
FACSCalibur (488- and 635-nm excitation), although they
should be the same independent of the instrument. This
difference is most likely due to the better signal-to-noise
ratio of the donor fluorescence because the 532-nm exci-
tation is more suited for the donors than is the 488-nm
excitation.
The last factor we investigated was the efr coefficient
(Table 4), and the largest differences occurred in this data-
set. The efr coefficients of Alexa555-containing dye pairs
Acceptors
FACSDiVa
Cy5 Alexa633 Alexa647
8.5 3.7 7.1
4.2 1.3 3.3
6.0 1.3 3.1
0.7 0.2 0.4

154

HORVATH ET AL.
were higher than those of the Alexa546-containing pairs,
which should have been the other way if only the excita-
tion spectra had been considered (Fig. 1). However, the
values with Alexa633 were (almost) the same on both cyt-
ometers, and on FACSDiVa the Alexa546-Alexa647 pair
had a higher efr value than did the Alexa555-Alexa647
pair. The efr values for the acceptors tested increased in
the order Alexa633, Alexa647, and Cy5 independent of
the donor on both cytometers. Therefore, when consider-
ing the efr values, the Alexa555-Cy5 pair seems to be the
best choice for FCET measurements.
The three instrument-dependent parameters were also
determined for the Alexa546-Alexa647 dye pair on the
FACSArray. The normalized fluorescence intensities were
5,356 and 20,852 (with background intensities 83 and 11)
for Alexa546 (LD 5 4.05) and Alexa647 (LA 5 4.54),
respectively. These values were even larger than those
obtained with FACSDiVa, probably due to the cuvette sys-
tem. The normalized FRET efficiency value was 0.352,
which was larger than those for FACSCalibur and FACS-
DiVa. The efr coefficient, determined as described in Mate-
rials and Methods, was the same (3.3) as that for the FACS-
DiVa, which can be explained by the similar laser lines
and detection optics.
DISCUSSION
In our study, we selected seven recently developed
fluorescent dyes, Cy3, Alexa546, Alexa555, and Alexa568
(FRET donors) and Cy5, Alexa633, and Alexa647 (FRET
acceptors), for flow cytometric energy transfer measure-
ments. The measurements were carried out on two cyt-
ometers, FACSCalibur and FACSDiVa, and the Alexa546-
Alexa647 dye pair was also tested on a FACSArray, with
excitation and detection characteristics similar to those
used for the FACSDiVa. To elucidate the comparison of the
fluorophores and cytometers, we characterized and mea-
sured four parameters that influence FCET measurements:
normalized fluorescence intensity, overlap integral, nor-
malized FRET efficiency, and the efr coefficient.
If we compare all datasets obtained on the three cyt-
ometers, the most significant feature is the pronounced
fluorescence intensity (and signal-to-noise ratio) with the
532-nm donor excitation, which is not surprising in view
of their excitation spectrum. However, the normalized
fluorescence intensities measured with different flow cyt-
ometers cannot really be compared even if the detector
and laser energy profiles are known because of the differ-
ent excitation and light harvesting conditions. One might
also note large differences between intensities due to the
different digitization schemes of the instruments, e.g., the
FACSCalibur uses 10-bit analog-to-digital conversion,
whereas the FACSDiVa, for pulse peak area, uses 18-bit
analog-to-digital conversion. Therefore, to increase the
sensitivity of FCET measurements, we can state that it is
not enough to use fluorophores that emit in the red region
of the visible spectrum (where the amount of autofluores-
cence is smaller) as was previously described (26), but the
flow cytometer has to be also equipped with lasers that
provide optimal excitation of these fluorophores.
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The difference in signal-to-noise ratio also had a great
effect on the FRET efficiency values. Signal-to-noise ratio
and FRET efficiency were higher on the FACSDiVa than on
the FACSCalibur for all tested dye pairs, although in princi-
ple the FRET efficiency values should be independent of
the instrument. This discrepancy challenges the compar-
ability of FRET results measured at the different research
centers and/or on diverse equipment. In general, FRET
efficiencies (even the distribution histograms) should be
quite similar if measured on the same instrument or the
same type of instrument, and the differences that arise
can be attributed to the biological variance of the samples.
This argument is probably also valid for the instruments
with similar excitation laser lines, optical filters, and
detectors. However, the change in the excitation wave-
length of the donor can seriously influence its quantum
yield (this physical parameter is usually not independent
of the wavelength, except in special cases), which in turn
has a large effect, via the F€
orster distance, on the value of
FRET efficiency. A more ideal excitation wavelength also
increases the donor signals and highly narrows the instru-
ment-dependent variance of the FRET efficiency distribu-
tion histograms, which can manifest in altered mean FRET
efficiencies. Thus, a feasible strategy to match data from
different laboratories should at least include the compari-
son of fluorescence intensities and normalized FRET effi-
ciencies and, if possible, quantum yield data of the fluoro-
phores in use.
The JDA and efr coefficient values presented in this
report may be useful in other experiments. Because the
overlap integrals were calculated from known excitation
and emission spectra of the dyes, these values can be used
to calculate the F€orster distance in the knowledge of the
donor quantum yield. The only problem may be the inser-
tion of k2, which in special cases can be estimated if
enough evidence is supplied. The most important feature
of the efr coefficient is that these values are transitive
between the same types of cytometers and can be used
for FCET measurements on any cytometer with the same
optical setup (this mainly stands for the commercial con-
figurations of FACSCalibur and FACSArray). These coeffi-
cients can also be derived from the excitation spectrum of
the fluorophores, but they are not so reliable because of
the small signal-to-noise ratio of the fluorescence of accep-
tor dyes at the donor excitation wavelength. It should be
noted that the efr coefficients obtained in this work and
those derived from the spectra are different, and the ones
presented in this work are more valid for measurements
on biological systems.
During the analysis of the results, two donor dyes (Cy3
and Alexa568) were left out from thorough investigation,
but they certainly can be considered with different cyt-
ometers and in other applications. Despite its large popu-
larity in genomic research, we recommend the use of Cy3
as a FRET donor for protein studies only if the expression
level of the molecule in question is high. However, the
Cy3-Cy5 dye pair is an ideal candidate for microscopic
acceptor photobleaching FRET experiments (33,34)
because Cy5 is highly susceptible to photobleaching, and

FLUOROPHORES FOR FLOW CYTOMETRIC FRET
with the available detection filters and mercury arc lamps
or 543-nm laser excitation Cy3 is very practical. The
strong argument for entirely excluding the Alexa568 dye
was its insufficient excitability with the 488- and 532-nm
lasers of our cytometers. However, if a cytometer is
equipped with a laser light source that has a characteristic
emission line closer to the maximum of the excitation
spectrum of the dye (e.g., the 543-nm line of a HeNe laser
or the 568-nm line of an Ar/Kr laser), then Alexa568
would be a far superior FRET donor fluorophore. Similar
lasers are already used in confocal microscopes, so the
Alexa568-Alexa647 pair (see JDA values listed in Table 2)
might be the most suitable FRET pair for intensity-based
FRET microscopy (4).
The two remaining donor fluorophores, Alexa546 and
Alexa555, were quite similar in every aspect of our investi-
gation, but Alexa555 was superior in most cases. In our
model system of highly expressed surface antigens, differ-
ences in signal-to-noise ratios of donor fluorescence were
not prominent with these two dyes, but the low fluores-
cence signal of the donor dye on a low-expressed antigen
can unfavorably influence the variance of FRET distribu-
tion histograms and seriously limit the sensitivity of FRET
measurements. Thus the most important parameter in the
case of a donor dye is its fluorescence intensity and,
hence, signal-to-noise ratio in the donor fluorescence
channel, which makes Alexa546 a better partner for FCET
experiments. (Here the higher normalized fluorescence
intensity can only be attributed to the better quantum
yield of Alexa546.) In the case of an acceptor dye, the
degree of spectral crosstalk to the donor fluorescence
channel and the overlap integral is more important. In this
respect, Cy5 seemed to be the best choice and Alexa633
the worst choice as a FRET acceptor and Alexa647 is very
similar to Cy5. The only reason we think Cy5 should be
considered as a less favorable fluorophore is its low quan-
tum yield and weak photostability, which have to be taken
into account if we want to expand our observations over
microscopic and fluorometric studies.
After analyzing the relevant four parameters (normal-
ized fluorescence intensity, Inorm; overlap integral, JDA;
normalized energy transfer efficiency, Enorm; and efr coef-
ficient), we found that the Alexa555-Cy5 and Alexa546-
Alexa647 pairs have the best characteristics. The
Alexa555-Cy5 pair exhibits the highest overlap integral
and FRET efficiency, whereas the Alexa546-Alexa647 pair
has the highest fluorescence intensity. Because the most
important parameter in fluorescence measurements is the
excitability and detectability of the fluorescent dye, the
Alexa546-Alexa647 pair is the most suitable fluorophore
pair for FCET experiments. In contrast, in some micro-
scopic techniques the photodestructibility of the fluoro-
phores is also important, so the Alexa555-Cy5 pair (from
the set of dyes we tested in this study) is the best choice
for acceptor photobleaching FRET measurements (9).
Unfortunately, we could not test flow cytometers other
than those from Becton Dickinson, but we have assessed
other cytometers from different manufacturers available
on the market; for a thorough list, see chapter 8 in Shapiro
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155
(32). We found that the FC500 (Beckman Coulter, Fullerton,
CA, USA) and the CyAn (DakoCytomation, Fort Collins, Col-
orado, USA) have characteristics similar to the FACSCalibur;
they share the same light sources, detection filters, and cuv-
ette system, so the results presented in this report are pre-
sumably valid for these two instruments and for two other
instruments from Becton Dickinson, the LSR II and FACS-
Aria. There is also the CyFlow instrument (Partec GmbH,
M€unster, Germany), which can be equipped with a whole
range of diode lasers, so it is possible to build a system that
is even more suitable (regarding optics) for FCET studies
than the commercially available setups we investigated.
FACSArray, a relatively cheap and incredibly fast instru-
ment, seems to be a great asset for large-scale FCET mea-
surements because it combines flow cytometric analysis
with FRET to yield a powerful, high-throughput assay for
the detection of molecular associations in intact cells.
Some other sets of fluorophores were also discarded
from this report, namely the phycobilin proteins, variants
of the green fluorescent protein (GFP), and quantum dots.
The phycobilin proteins, R-phycoerythrin and allophy-
cocyanin, and their tandem conjugate derivatives are
very popular in immunofluorescence studies because
their exceptional excitability (
2 million 1/M* cm for R-
phycoerythrin) allows for the detection of hundreds of
antigens (35). However, the size of these proteins is
comparable to that of antibodies and thus renders them
inaccurate or even impractical for detecting small mole-
cular distances. Another consequence of their size is
that they move relatively slowly and cannot rotate as
freely as small fluorescent molecules, which has a tre-
mendous effect on their orientational freedom. This
inability manifests in such k2 values, where the range of
FRET efficiency is impractical (36).
When fluorophores conjugated to antibodies are used
as donor-acceptor pairs in FRET studies on live cells, these
approaches are often restricted to the extracellular part of
molecules and depend on the availability of appropriate
antibodies. The recent development of GFP variants suita-
ble for FRET has expanded the utility of this methodology
by permitting the study of intracellular and extracellular
processes (37–39). However, despite their known advan-
tages and recently gained importance, there are several
real disadvantages that render them difficult to use in
FRET studies. Combining GFP variants to form good FRET
pairs takes some amount of prudence because the excita-
tion and detection ranges are usually quite close so that
spectral crosstalk may hinder the accurate determination
of FRET efficiencies. These difficulties caused by spectral
overlap can be overcome by the detection of the entire
spectrum and mathematically unmixing the components
(40,41). Another limitation is that most available laser
lines are not optimal for excitation, and the GFP variants
mostly cover the lower part of the visible spectrum,
where autofluorescence greatly limits the sensitivity.
Nonetheless, some new variants emitting in the red region
(DsRed) may be successfully applied for FRET studies
(42). A great disadvantage is the need for cotransfection of
two proteins. This difficulty can be circumvented by

156

HORVATH ET AL.
studying the interactions of GFP variants with homotrans-
fer, which occurs between fluorescent molecules of the
same kind and manifests in the decrease of fluorescence
anisotropy. This method has recently been applied to
microscopy (43) and with little investment and few altera-
tions can be implemented for flow cytometry.
The use of quantum dots in biomedical studies is an
emerging methodology that should be considered in rela-
tion to FRET measurements. The main advantages of quan-
tum dots are the wide excitation spectra, narrow and sym-
metrical emission spectra, good quantum yield, and
photostability (44,45). However, some of these para-
meters may manifest as disadvantages in FRET studies.
Due to the extremely broad excitation spectrum, quantum
dots cannot be used as FRET acceptors because the
enhancement on the acceptor side would be small and
thus compromise the accuracy of FRET measurements.
When quantum dots are used as donors, the narrow emis-
sion spectra would decrease the overlap integral with the
acceptor dye, thus decreasing the R0 value for the donor-
acceptor pair. Another disadvantage is their size: although
the inner core is relatively small (2–5 nm), the coating for
protection and biological linking increases the size to 15
to 20 nm, when FRET measurements become impractical.
In summary, to study associations of membrane pro-
teins in cells in suspension, we have established the para-
meters of normalized fluorescence intensity, overlap inte-
gral, normalized FRET efficiency, and efr coefficient as cru-
cial for FCET measurements and found that the optimal
donor-acceptor pairs are Alexa546-Alexa647 and
Alexa555-Cy5 in the three flow cytometers tested, with
the latter also practical in microscopic acceptor photo-
bleaching experiments. The use of high-throughput FRET
measurements is greatly facilitated by the FACSArray flow
cytometer. If the optical parameters of the flow cytometer
change (e.g., a new laser line is introduced), a similar eva-
luation procedure as described in this report can be used
to determine the optimal donor-acceptor pair for the
given instrument setup.
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