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Making fluorescent microscopy less finicky

10/19/2010
Suzanne E. Winter

Scientists are improving the stability and brightness of many fluorescent probes, enabling previously unimaginable experiments. Suzanne Winter examines how these changes are leading to new cellular insights.

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Randall Goldsmith, a postdoctoral researcher at Stanford University, pauses as he begins to describe his current research. "Well,” he sighs, “fluorescent proteins are rather finicky." Goldsmith’s delicate critique of one of molecular biology’s favorite tools speaks volumes. Fluorescent probes can photobleach, quickly disappearing with the target molecule into darkness. They can also bind indiscriminately to deliver nonspecific signals and, depending on the probe, they can attach themselves to an imaging device, changing the native kinetics of the unbound target molecule or removing it from the imaging plane. But when they do behave as intended, fluorescent molecules can yield breathtaking results.

Goldsmith and Moerner’s ABEL trap stabilizes a fluorescent probe to allow for extended observation; here, a single lambda DNA molecule is recorded in the ABLE trap. Source: Moerner lab website.

Tweaking fluorescent probes to improve performance has become a science in itself for many researchers. “The physical process is cool: you absorb a photon, and then you get one back, but it’s a different color from the one you put in,” says Bruce Armitage, professor at Carnegie Mellon University (CMU), and one of a growing number of scientists enhancing the current palette of fluorescent molecules. “That captivated me from the time I was an undergraduate.”

Stabilizing the lazy octopus

W.E. Moerner speaks of single-molecule imaging like a precious art form—no surprise if one considers that his Stanford University lab has been developing single-molecule spectroscopy, super-resolution imaging, trapping, and nanophotonics approaches over the past 20 years. “By watching individual molecules, we can see behavior that’s hidden when you do ensemble averaging,” he says. “In biological systems, each object that you might be interested in, an enzyme or a special protein, for example, is different; if you measure many of them at the same time, you essentially obscure the behavior of the individual.”

In order to understand the roles that individual molecules play, researchers in Moerner’s lab try to observe single molecules in solution, but are at the whim of the molecule’s Brownian motion. The molecule ebbs with any thermal agitation in the fluid and is apt to flow right out of the focused laser beam, which limits the observation period of the molecule. Although methods to stabilize this motion exist, many of them induce conformational changes in the molecule, altering its native kinetic or physical attributes.

To combat inadvertent conformation changes, Adam Cohen, a former Moerner postdoc now at Harvard University, developed the anti-Brownian electrokinetic (ABEL) trap, a machine that applies an electrokinetic force to the molecules under observation. When Brownian motion or thermal fluctuations interact with the targeted biomolecule, such as kicking it to the left of the observation plane, the trap registers this change and produces a kick of equal force and opposite direction to reposition the biomolecule in the center of the observation plane.

“It’s a closed-loop feedback device,” explains Moerner. “It allows us to hold a biomolecule in solution for a long time until the fluorophores of the molecule photobleach.” This allows his team to categorize the single molecules in their native state without suffering any of the conformational changes associated with other stabilizing methods.

Goldsmith, who is currently a postdoc in Moerner’s lab, describes the trap a bit differently. “[A single molecule] is like an octopus,” he says. “If you go to the aquarium to watch the octopus, it’s sort of going off in all random directions, and you have the trainer nearby just sort of pulling or pushing the octopus back into the center so that all the cameras in the aquarium can see it.”

Recently, Goldsmith and Moerner chronicled their modifications to the ABEL trap in a paper published in Nature Chemistry (1), using it to observe and document the photophysics of allophycocyanin (APC), a single fluorescent protein. “We wanted to use it on a real biomolecule, a small protein, to demonstrate what can be done with this trap,” says Moerner.

Before they could accomplish this, Goldsmith had to retool the trap because some fluorescent proteins, APC included, are quite sticky in a microfluidic environment, which interferes with the ABEL trap’s ability to relocate the molecule to the center of the observation plane. To address this, Goldsmith applied a series of charged polymeric surface coatings, called polyelectrolyte multilayers, to repel the fluorescent protein from the surface while it experiences the electro-osmotic forces of the trap.

With their modifications, Goldsmith and Moerner tested the imaging capabilities of the ABEL trap to see if they could maintain the native conformation and behavior of the molecule under observation, or would they still perturb the octopus? “Based on the evidence that we’ve gathered and the calculations that we’ve done in this paper, we don’t think we have perturbed it.”

Their data suggests that repeated excitations of fluorescent molecules lead to changes in their radiative lifetime, a value previously assumed to be static. “As much as we want these proteins to be impartial observers,” says Goldsmith, “people need to be aware that the proteins they’re using also interact and have individual behaviors. One of those behaviors is a non-static radiative lifetime.”

Photographing before photobleaching

In addition to radiative lifetime changes, fluorescent molecules only have a certain luminous lifespan, after which they photobleach and cease to emit light. Clearly, this poses a problem for researchers, who must base their observations around the temporal caprice of the fluorescent tags associated with their target molecules. “If you’re taking measurements on a molecule until, basically, it’s chemically exploding as it photobleaches, then you can’t repeat the measurement on the same molecule,” sums up Goldsmith. “So how do you convince yourself and your peers that you’re doing good science?”

By using DNA as a scaffold for numerous reversibly-binding fluorescent molecules, Armitage is working to create long-lasting and self-replenishing probes. Source: Armitage lab website.

At CMU, Armitage’s lab is developing dyes that bind reversibly to the target protein and only fluoresce when bound, eliminating nonspecific background noise and reducing the threat posed by photobleaching. “It’s all based on concentration,” says Armitage. “Most of the dyes will bind to the protein at a low concentration, and so as long as the concentration of dyes remains ten or so times above the minimum binding concentration, the protein will always have a dye bound to it and anything that’s not bound remains dark.” Instead of targets binding irreversibly to a tag that will eventually photobleach and cause them to become lost, the high concentration of reversibly binding fluorescent molecules promotes exchange between bleached dyes and fresh dyes that, up until that point, have been floating darkly in the flask or cell. This extends the observation period by providing a renewable source of fluorescence.

Armitage’s group is also working on methods to extend fluorescence observation times while enhancing the brightness of the fluorescent molecules. In traditional fluorescence practice, like with green fluorescent protein (GFP), one fluorescent dye would be attached to one protein; therefore, the observation ends when the dye photobleaches. To go beyond the photobleaching limitation of fluorescence proteins, Armitage is building fluorescent DNA nanostructures, called DNA nanotags, which can label proteins and RNA ten to fifteen times brighter than conventionally available fluorescent dyes. “We’re attaching ten, twenty, thirty dyes to each piece of DNA, and the DNA becomes a label that we attach to other things,” says Armitage. “The DNA is like a scaffold that lets us assemble these fluorescent dyes in one location, and then it becomes extremely bright because there are so many of these dyes.”

Another issue on Armitage’s mind is that certain fluorescent dyes are easier to produce than others. For example, researchers are increasingly interested in tracking fluorescent molecules in deep tissue samples, where a longer wavelength enables stronger observations at a greater depth. Despite this high demand, there are limited quantities of fluorescent dyes with long wavelengths because they are difficult to produce. “The longer wavelengths are much harder,” says Armitage. “They tend to photobleach faster and there are more synthetic steps usually required to make them, as they tend to be larger molecules.” Armitage and his lab are hoping to improve the luminescence time of these dyes and simplify their creation in order to make a wide spectrum of colors available for the greater research community.

Fluorescent future

Goldsmith and Armitage are hoping to change the field of fluorescent imaging for the better. Goldsmith’s modified ABEL trap, with the associated change-point-finding algorithm and Goldsmith's original time-order clustering method, promises to lead to further super resolution kinetic classification of fluorescent molecules. “Whenever I present this material,” says Goldsmith, in reference to the trap and combined algorithms, “everyone gets really excited. You can look at [ABEL trap data] and say, ‘Aha, there are plateaus there; wouldn’t it be nice if I had an automated way of identifying them?’ Poof, you have one.”

For his part, Armitage is also enthusiastic about the future of single-molecule imaging. “The biggest advances have been trying to extend fluorescence to single molecule measurements, and that’s where our bright labels will come in handy once the technology is mature,” says Armitage. “You’ll be able to see a single molecule in a much shorter imaging time if you have much brighter fluorescence.”

References

(1) Goldsmith, R.H. and W.E. Moerner. 2010. Watching conformational- and photodynamics of single fluorescent proteins in solution. Nature Chemistry 2: 179-186.