The Optical Trap

OPTICAL TRAPPING

Optical traps exploit the momentum of laser light to manipulate individual objects – not just beads, but also cells, organelles, and even atoms. After years of work, the Bell Labs group found they could hold a clear bead in place with a single, tightly focused laser beam, a surprisingly simple configuration now known as optical tweezers. If the bead starts to drift away, the laser light is deflected, and the particle is pushed in the opposite direction, back towards the focus.

The result is that the bead is held gently in place, as if by tiny springs. When the experimenter moves the light beam, the bead follows, so optical tweezers can move objects around like their nonoptical namesake. Moreover, unlike alternatives such as glass fibers or micropipettes, optical tweezers can reach into a cell without disrupting it. They never break, and "cleaning" them is as simple as turning off the light.

Moving beyond traditional tweezers, researchers can also measure and change how hard the trap pulls on the bead. First, they calibrate the trap's "stiffness." Then, by measuring precisely how far off-center the bead is, they can deduce the optical restoring force being applied. The forces, typically on the order of tens of piconewtons, are similar to those exerted by the molecular motors that power muscle and actively transport material within cells. Finally, by incorporating the measurement into a feedback loop, the experimenters can tune the applied force. That makes optical traps particularly useful in molecular motor research, as it becomes possible to ask, how hard must I tug on a protein to make it stop moving?

Says Stanford biochemist James Spudich, "Tremendous numbers of things have been learned about the motors using this technology."

MOLECULAR MOTORS

When Spudich began using optical tweezers in the early 1990s, he had been engaged for nearly a decade in what he calls a "lively argument" about how myosin moves. Spudich believed this molecular motor took tiny steps, only about 10 nm long. Others favored a step size an order-of-magnitude larger. Both conclusions were based on a system of polystyrene beads coated with purified myosin, which Spudich and Michael Sheetz (now at Columbia University) had developed a decade earlier. The observations were indirect, however, and supported both hypotheses.

Now Spudich's group had a way to answer the question directly. In collaboration with Robert Simmons of King's College London, at Stanford on sabbatical, and Chu, who had arrived from Bell Laboratories in 1987, they built an optical tweezers apparatus that could resolve the steps of a myosin-coated bead to within 10 nm. They applied their beads to actin-coated slides, gently tugged on them with the tweezers, and watched them march along the filament, just as they do in vivo.

"We had to see a small step in a background of Brownian motion noise," Spudich recalls. "If we were right, the step size was going to be buried in that noise." Spudich estimates the team spent about $70,000 assembling the apparatus, but it wouldn't work until graduate student Jeff Finer bought "a few dollars worth of cardboard" at the university art store to block air currents. Finally, they had their answer: the beads moved in discrete steps of about 10 nm. "I don't think there's been any argument since," Spudich adds.

SINGLE MOLECULE EXPERIMENTS

For all their power, optical tweezers are "just one in an arsenal of tools" for single-molecule work, notes Block. Optical tweezers are most useful for moderate forces, in the range of 1 to 100 piconewtons, says Carlos Bustamante of the University of California, Berkeley. Stronger forces are better studied using atomic-force microscopy or micropipettes, he says, while weaker forces are best studied using magnetic beads in a magnetic field.

There are other tools, too. "With traps you can manipulate things, you can apply forces to them, but you can't actually visualize simultaneously what's happening at the protein level in terms of conformational changes," Block points out. For that, researchers have used tools that can track, with nanometer precision, the movement of fluorophore-tagged protein molecules.

Block's team, and others, have combined fluorescent probes with optical traps – an engineering feat that requires detecting minute flashes of fluorescence in the presence of the intense light used for trapping. "It's now possible to do experiments that were considered pipe-dreams only a decade ago," says Block. Bustamante, who has combined fluorescent probes with magnetic tweezers, says, "This is a very important development."

CONTINUING EVOLUTION

Naturally these new systems don't spell the end of the trap's long development. Though a basic commercial optical tweezers set-up can be purchased for around $10,000, a cutting-edge system, capable of applying precise forces to multiple particles and precisely measuring their positions, costs $150,000 or more, just for parts. Perhaps more importantly, the most advanced research still demands expertise in optics and electronics, as well as biology.

For almost a decade, researchers have been building tweezers with two traps, either by using two lasers, or by rapidly switching a single laser between two positions. Recently they have been exploring more complex configurations.

David Grier of New York University, for example, devised a holographic scheme for creating arrays of many traps. Jesper Glückstad of RISO, in Denmark, uses a different concept to form a sort of optical petri dish, which he and his collaborators have used to explore how the propagation of yeast cells changes when they are surrounded by cells of a different species. These multitrap tools could also explore the cell-cell interactions involved in tumor formation or stem cell differentiation.

Other groups have devised ways to apply torque in optical traps, by harnessing light's angular momentum. Traditional optical traps exert linear forces, but not torques. Thus to manipulate topoisomerases and helicases, researchers had to use magnetic beads instead of traps. Using the new schemes, which are still in early development, the torque exerted when light scatters from a rodlike object can be measured and even controlled, just as linear force can.

Block's group has spent years steadily improving the position sensitivity of its instruments by eliminating noise and improving detector precision, using schemes considerably more sophisticated (and expensive) than Finer's cardboard. Their patience is now being rewarded, with systems that can resolve a bead's position to within a fraction of a nanometer.

That resolving power gives optical traps the power to address questions like the conformational changes hemoglobin undergoes upon binding oxygen, and how polymerases work. Block's group has used a system with two independent traps to study transcription. By gently pulling apart two beads – one attached to DNA and another to RNA polymerase – they tracked the transcriptional motion of the polymerase along the DNA with subnanometer precision, observing long pauses they ascribed to backtracking and error correction.¹

GOING IN VIVO

John Kendrick-Jones, a myosin expert from Cambridge University, says watching individual protein molecules in action is a "fantastic" achievement. But, he cautions, "the results you get have to be taken with a pinch of salt, because of course you are not mimicking the cellular environment." He adds: "Optical tweezers does actually lend itself to some extent to start to look at events within cells."

Controlled manipulation of the cell's internal structure will be challenging, but exerting some force is not difficult; Ashkin first pulled on organelles in live cells in 1989.² "If you can see a structure, you can apply a force to it," says Grier, "provided it's not embedded and not swimming too hard." The irregular geometries of cellular organelles makes quantitative measurements of forces and displacements difficult, but Sheetz says the bigger challenge is that many structures are embedded in the cytoskeleton. "The force required to break an actin filament is 400 piconewtons. That's impossible with optical tweezers."

Manipulating entire cells is easier. Ellen Townes-Anderson of the University of Medicine and Dentistry of New Jersey has been using optical tweezers to construct networks of retinal neurons. Even a basic commercial instrument allows more precise placement of the cells than micropipettes, she says. "We were able to do it right off the bat." The main challenge has been developing a substrate from which cells can be gently plucked.

The resulting circuits allow Townes-Anderson to explore the rules that govern how adult nerve cells grow and connect, which could be important for developing implants. She hopes also to create controlled interconnections between nerve cells and electronic chips.

In 1991 Chu wrote, "It is a personally gratifying example of the unity and synergism of science that seemingly esoteric work in atomic physics is having impact in chemistry, biology, and may even give us new methods to study fundamental processes of life."³ The optical trap has now turned that corner.

"Finally we are going to start learning some new biology." says Bustamante. Kendrick-Jones agrees: "One hopes it will become more and more a biological tool: that the biophysicists will move in with the cell biologists, and hand in hand, together we'll be able to explore within the cell. I think that's the great, exciting thing." We've just begun to scratch the surface," says Block.

Optical Trapping Milestones

1958

Invention of laser