Hank Hogan, firstname.lastname@example.org
When it comes to laser sources, three are definitely a crowd. That is particularly true
when trying to align multiple beams to illuminate the same confocal volume. So researchers
at the National University of Singapore used just one beam and multiple fluorophores
in demonstrating that simultaneous multicolor fluorescence cross-correlation spectroscopy
could detect interactions among three molecular partners.
These experiments were necessary for achieving
the final objective of elucidating the space and time interaction patterns of signal
transduction pathways in vivo, according to Thorsten Wohland, assistant professor
of chemistry at the university and research team leader.
Using the fluorescence correlation spectroscopy technique, researchers measure fluorescence intensity fluctuations in a confined area. From these changes they extract such parameters as diffusion
coefficients and chemical rate constants. When following multiple chemical reactants
and products that have molecular masses within a factor of four or so of each other,
they have to use multiple labels. Each component will then have its own emission
signature that can be tracked in a separate detector channel.
In the past, exciting these multiple
fluorophores has meant that either two or three distinct beams must be used or two-photon
excitation employed. The first choice entails a difficult alignment while the second
can be done only with an expensive femtosecond laser and at the cost of lower emission
In these two setups, a laser and a combination of fluorophores perform multicolor fluorescence
cross-correlation spectroscopy. Either setup eliminates the need to align multiple
beams or to use an expensive femtosecond laser. On the left is a conventional setup,
with the signal split by dichroic mirrors into red, yellow and green channels directed
into separate avalanche photodiode detectors. On the right, the signal is split
into three channels with a dispersive element. Images courtesy of Thorsten Wohland
and the National University of Singapore.
In contrast, single-wavelength fluorescent
cross-correlation spectroscopy uses a single beam, avoiding problems of alignment,
cost and low emission. It does require that the fluorophores have similar excitation
spectra — so that one beam can excite all of them — but spectrally different
emission characteristics. The method also demands limited crosstalk among the fluorophores,
so that the emission of one doesn’t affect the emission of another. As a result,
the fluorophores should have a large Stokes shift, or spectral separation between
excitation and emission peaks.
Wohland noted that this combination
of characteristics has kept single-wavelength fluorescent cross-correlation spectroscopy,
which was first demonstrated years ago, from widespread use. In commonly used small
organic dyes, for instance, crosstalk is very high. The situation, however, has
The above graphs show the strength of cross-correlation in the spectroscopy
signal, assuming a red-emitting receptor (red H-shaped structures) with a binding
site each for a yellow- (blocks) and green- (solid circles) emitting ligand. Researchers
can determine which interactions take place among the three molecules by calculating
the different cross-correlations in the measured data, indicated by the arrows
pointing to the surface.
“With the advent of quantum dots,
mega-Stokes dyes and, very recently, the fluorescent proteins with large Stokes
shift, this technique becomes now much more interesting because of its ease of use
and the lower cost,” Wohland said.
In a series of experiments, the group
used a single laser to excite three molecules. One was a green ligand consisting
of biotin labeled with fluorescein. Another was a yellow ligand composed of biotin
labeled with R-phycoerythrin. The receptor was red, created by labeling R-phycoerythrin-streptavidin
with Alexa Fluor. Because the receptor had four binding sites, any combination from
zero to four of the two biotin ligands could attach to a single receptor.
In their study, the researchers used
an argon-ion laser from Lasos Lasertechnik GmbH of Jena, Germany, employing a 488-nm
filter from Chroma Technology Corp. of Rockingham, Vt., to transmit only one of
the two laser lines. They sent this filtered beam through an Olympus microscope
and focused it onto a sample solution. They collected the emission with the same
objectives, employing a dichroic mirror from Omega Optical Inc. of Brattleboro,
Vt., to separate the fluorescence from the scattering of the laser beam. Two more
dichroic mirrors, also from Omega and operating at 560 and 630 nm, split the emission
into green, yellow and red detection channels.
After passing the emitted light through
various optics designed to focus and filter it, the investigators used PerkinElmer
avalanche photodiodes to detect the signal. They used three PCs to do pair-wise
cross correlations, looking for green and red, yellow and red, and green and yellow
Wohland said that dispersive elements
in the detection pathways and the possible use of CCDs allow for the easy adjustment
of wavelength channels as compared with other approaches. Such improvements
might mean more detection channels rather than a change in detection limits. “The
advances in setup technology concern more the wavelength resolution and thus the
number of channels achievable,” he said.
The researchers chose dyes that had
largely different Stokes shifts, minimizing crosstalk. They also picked ones with
similar excitation characteristics so that they would respond similarly to the laser.
They then chose the dichroic elements and filters to match the maximum emission
With their setup, they measured the
effect of introducing various concentrations of green- and yellow-labeled biotin,
employing negative controls in which only one was allowed to bind to the receptor.
As the molecules drifted into and out of the focal volume, the fluorescence fluctuated
because of the drift, transitions between various states and interactions between
the reactants. After algorithmically processing the raw measurements, the researchers
accurately determined the interaction among the three differently labeled species
as well as the stoichiometry, or quantitative reactant and product relationships,
of those interactions.
Wohland noted that these results were
as expected, largely because of the high affinity between biotin and streptavidin.
He expects to see the technique applied to other nontest situations because it can
offer information that is difficult to determine by other methods. When looking
at interactions that vary over time, for example, the method can resolve patterns
and show in which sequence up to three different molecules bind to one another.
There are, of course, some shortcomings
to the approach. Going to a greater number of interactions than three would require
a larger number of fluorophores, leading to narrower bandpasses in the optical path
and potentially more crosstalk. Impurities, which can arise because of inactive
or unlabeled receptors or ligands, reduce the difference between positive and negative
controls and so decrease sensitivity. That could limit the applicability of the
method. A consequence of the first two limitations is that dissociation constants
are hard to determine accurately.
However, for Wohland, the positives
outweigh the negatives. With the right labels now available, only the right labeling
approach is needed. “The real challenge now is to use these dyes for in vivo
experiments,” he said.
Contact: Thorsten Wohland, National University of Singapore; e-mail: email@example.com.