Multiplexed FRET to Image Multiple Signaling Events in Live Cells
David M. Grant,* Wei Zhang,yEwan J. McGhee,zTom D. Bunney,yClifford B. Talbot,zSunil Kumar,*
Ian Munro,zChristopher Dunsby,zMark A. A. Neil,zMatilda Katan,yand Paul M. W. Frenchz
*Chemical Biology Centre, Imperial College London, United Kingdom;yCancer Research UK Centre for Cell and Molecular Biology,
Institute of Cancer Research, London, United Kingdom; andzDepartment of Physics, Imperial College London, United Kingdom
energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric
spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence
for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon
and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of
multiple FRETsensors withinthe same cell. Furthermore, sinceFLIMdoesnot requireequal stoichiometriesofdonor andacceptor,
this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.
We report what to our knowledge is a novel approach for simultaneous imaging of two different Fo ¨rster resonance
Received for publication 3 June 2008 and in final form 19 August 2008.
Address reprint requests and inquiries to David M Grant, Tel.: 0207-594-1278; E-mail: email@example.com.
The signaling networks that underlie complex cellular pro-
cesses such as cell survival, proliferation and differentiation
comprise a vast number of different proteins and secondary
cell behavior. Understanding the mechanisms behind different
correlated in time and space. To this end Fo ¨rster resonance
energy transfer (FRET), the nonradiative transfer of energy
from an excited state fluorophore (the donor) to a spatially
colocalized chromophore (the acceptor), provides a means to
utilized only a single donor-acceptor pair because the ability
to image multiple FRET pairswithina single cell—andthus to
correlate multiple signaling events—has been limited by the
particularly thegeneticallyexpressedfluorescent proteins.Pre-
vious strategies to overcome this problem have included the
this approach could only be applied to study of interactions
where the binding partner was the same for both donor-labeled
species. The recent growth in red and orange fluorescent pro-
teins has addressed this issue to some extent by increasing
thespectral bandwidthavail-able (3,4),although someof these
longer emitting proteins exhibit relatively low quantum eff-
to work well as FRET pairs (4) and the first multiplexed
and ECFP/EYFP, was recently described (5).
Ratiometric FRET measurements can, however, be compro-
mised by cross talk which can significantly limit the achievable
signal/noise ratio. Depending on the wavelength used to excite
the mOrange donor andthe filter usedto detect its emission, it is
can be minimized by exciting and detecting at longer wave-
lengths but only at the expense of increased direct excitation of
shifted donor channel. Together with the inevitable bleed-
through of donor fluorescence into the acceptor channel, this
cross talk can result in a high noise background against which it
lem of fluorescence from direct excitation of the first acceptor
being detected in the second donor channel, since both donors
can now be excited at a wavelength beyond the excitation spec-
Here,we report a method for imaging two FRET pairs within
a novel redprotein TagRFP was introduced (7) that has a higher
and whose fluorescence can easily be resolved from that of
EYFP. TagRFP can therefore serve as a suitable donor for
Editor: Michael Edidin.
? 2008 by the Biophysical Society
Biophysical Journal: Biophysical Letters
Biophysical Journal: Biophysical Letters L69
having the longest emission spectrum of all currently available
fluorescent proteins, is a possible choice. One reason why this
pair had not previously been utilized for FRET is that the low
quantum yield of mPlum makes it unsuitable for ratiometric
measurements. Our approach overcomes this problem by using
the TagRFP/mPlum pair. Unlike spectral ratiometric measure-
ment, FLIM only requires the donor fluorescence signal to be
spectral ratioing of an ECFP/Venus pair, we were hence able to
maximize the spectral separation of our chosen FRET pairs
while avoiding the issue of low sensitized emission from the
second (long wavelength) acceptor. In addition, since fluores-
cence lifetime is largely independent of fluorophore concen-
tration, FLIM-FRET does not require equal stoichiometries of
FRET as well as dual labeled biosensors.
Our multiplexed FRET instrument is built around an in-
verted Olympus microscope into which are coupled a contin-
and Venus fluorescence emission are resolved from TagRFP
two channels are imaged onto the same charge coupled device
mapped to changes in calcium concentration throughout the
cell. FLIM images of the TagRFP donor are acquired using a
high speed wide-field time-gated method (8).
with epidermal growth factor and spectral ratiometric imaging
was used to monitor changes in calcium flux using an ECFP/
Venus Cameleon sensor (YCAM 3.6). FLIM meanwhile was
Domain from C-Raf-Kinase (Raf-Ras Binding Domain) and
between the TagRFP/mPlum pair provides a sensitive means
and mPlum acceptor is indicated by a decrease in TagRFP
detect the fluorophores.
Fig. 3 shows ratiometric images of the Cameleon sensor at
images of Cameleon were acquired by exciting ECFP with a
continuous wave blue laser and resolving the fluorescence into
two spectral channels. For FLIM measurements an ultrafast
supercontinuum excitation source (Fianium, UK) was spectrally
filtered for TagRFP excitation. TagRFP fluorescence was imaged
ontoa GOI (gatedoptical intensifier (KentechInstruments, model
HRI) and time-gated fluorescence images were recorded on a
Hamamatsu ORCA-ER charge coupled device camera.
Multiplexed FRET microscope. Spectral ratiometric
results in a conformational change and FRET from ECFP to Venus.
Activation of mPlum labeled H-Ras (exchange of GDP for GTP
catalyzed by guanonucleotide exchange factor (GEF)) results in
recruitment of Tag-RFP labeled Raf-Ras Binding Domain to the
membrane and FRET between TagRFP/mPlum. (B) Absorption and
emission spectra of the 4 fluorophores. Shaded areas indicate
excitation and emission bands used for imaging multiplexed FRET.
Filters are given in the Supplementary Material, Data S1.
FRET constructs and spectral channels used. (A)
Biophysical Journal: Biophysical LettersL70
Biophysical Journal: Biophysical Letters
lifetime images of the second donor TagRFP. After stimulation
with epidermal growth factor, we observed a transient rise in
the plasma membrane, as evidenced by the shorter fluorescence
lifetime around the cell peripheries. Fig. 3 C shows mean
intensities in the CFP and Venus channels from a region in the
cytosol together with mean fluorescence lifetimes at the cell
intensities and TagRFP lifetime remained constant in cells not
stimulated by EGF.
One limitation of this method is that TagRFP was found to
FLIM images that could be acquired before the fluorophore
bleached. This meant that we were able to obtain fewer time
points in the time lapse sequence of Ras activation than were
mutant with greater photostability has just become available (9)
although these experiments were performed using a wide field
microscope, it would also be possible to implement optical
sectioning using a Nipkow disc confocal microscope, and so
In conclusion, we have developed a method for imaging
and calcium flux in live cells. Such a system holds promise for
elucidating the role of calcium signaling in Ras activation and
how the spatial and temporal modulation of calcium concentra-
sensors available to biologists continues to expand, we envisage
that using this approach it will become possible to explore the
interplay and interdependency of a host of cellular parameters.
To view all of the supplemental files associated with this
article, visit www.biophysj.org.
This work was supported by the UK Biotechnology and Biological
Sciences Research Council (BBSRC), Cancer Research UK, the European
Community (Framework VI Integrated Project ‘‘Integrated technologies for
in vivo molecular imaging’’ contract No. LSHG-CT-2003-503259), The
UK Medical Research Council and the Department of Trade and Industry.
D. M. Grant and S. Kumar acknowledge studentships from the Chemical
Biology Centre, Imperial College London, supported by the UK Engineer-
ing and Physical Sciences Research Council (EPSRC).
REFERENCES and FOOTNOTES
1. Jares-Erijman, E. A., and T. M. Jovin. 2006. Imaging molecular in-
teractions in living cells by FRET microscopy. Curr. Opin. Chem. Biol.
Ras isoforms simultaneously in a single cell. ChemBioChem. 6:78–85.
3. Shaner, N. C., R. E. Campbell, P. A. Steinbach, B. N. Giepmans, A. E.
Palmer, and R. Y. Tsien. 2004. Improved monomeric red, orange and
yellow fluorescent proteins derived from Discosoma. red fluorescent
protein. Nat. Biotechnol. 22:1567–1572.
4. Goedhart, J., J. E. Vermeer, M. J. Adjobo-Hermans, L. van Weeren, and
5. Piljic, A., and C. Schultz. 2008. Simultaneous recording of multiple
cellular events by FRET. ACS Chem. Biol. 3:156–160.
6. Ai, H. W., K. L. Hazelwood, M. W. Davidson, and R. E. Campbell.
2008. Fluorescent protein FRET pairs for ratiometric imaging of dual
biosensors. Nat. Methods. 5:401–403.
A. F. Fradkov, A. Gaintzeva, K. Lukyanov, S. Lukyanov, T. W. Gadella,
an extended fluorescence lifetime. Nat. Methods. 4:555–557.
8. Suhling, K., P. M. French, and D. Phillips. 2005. Time-resolved
fluorescence microscopy. Photochem. Photobiol. Sci. 4:13–22.
9. Shaner, N. C., M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L.
Hazelwood, M. W. Davidson, and R. Y. Tsien. 2008. Improving the
photostability of bright monomeric orange and red fluorescent proteins.
Nat. Methods. 5:545–551.
10. Grant, D. M., J. McGinty, E. McGhee, T. D. Bunney, D. M. Owen, C. B.
A. I. Magee, P. Courtney, M. Ka-tan, M. A. A. Neil, and P. M. W. French.
study of live cell signaling events. Opt. Express. 15:15656–15673.
activation. (A) Spectral ratiometric images of Cameleon at time
points shown (times in seconds). Epidermal growth factor was
added 10s from the start. (B) Lifetime maps of TagRFP at time
points shown (top row) and lifetime merged with intensity (bottom
row). (C) Intensity tracesin the Venus and ECFPspectralchannels
from a region of interest in the image and mean lifetime of TagRFP
from a region in the membrane. Details of cell culture and plasmid
construction are given in the Supplementary Material, Data S1.
Multiplexed FRET imaging of calcium flux and Ras
Biophysical Journal: Biophysical LettersL71
Biophysical Journal: Biophysical Letters