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 rates. 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 changed. 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 interactions. 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 wavelengths. 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: chmwt@nus.edu.sg.