Optical Trapping Milestones
When Art Ashkin, Steve Chu, and their colleagues at Bell Labs in Holmdel, NJ, first invented optical tweezers, they spent their days pushing around tiny, glass spheres. But it wasn't long after their 1986 discovery that they began to think about biology.
"We were trapping submicron particles of Tobacco mosaic virus," Ashkin says. "We left the samples under the microscope for a day or so, and then we discovered strange particles that seemed to be self-propelled." When they looked into the trap with a higher-quality microscope, they confirmed what the mysterious objects were. "They discovered bacteria, 350 years too late," jokes Howard Berg, who was then studying bacterial flagella at the Rowland Institute in Cambridge, Mass.
The tweezers' intense green light quickly killed the bacteria, a process Ashkin dubbed "opticution." But once the team switched to an infrared laser, the bacteria could be kept alive indefinitely, even reproducing in the trap. This innovation "pointed the way to the possibility that one could really study biological things" using optical tweezers, says Steven Block, who was then working in Berg's lab.
The Rowland researchers were hooked. "We got excited about this paper," Berg recalls. "That was on a Tuesday. The following day we got permission from [Rowland benefactor Edwin] Land to buy a laser, and by Friday we were up and doing experiments."
They first tethered a flagellum to a substrate, so that its action set the bacteria spinning, like a tail wagging a dog. They then paralyzed the flagellum and used optical tweezers to gently pull the bacteria sideways. By watching it return to position, they measured the elasticity of the specialized protein complex (the "hook") that couples the flagellum to the cell. Thus began the quantitative application of optical tweezers to biological systems.
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."
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.
The Father of the Optical trap
Art Ashkin is widely recognized as the father of optical tweezers. Working at Bell Labs, Ashkin took a keen interest in harnessing the forces that laser light exerts on atoms and particles. As early as 1970, he levitated micron-sized particles, and he soon built a stable trap using two beams intersecting each other head-on. In 1976 he proposed similar methods to trap atoms.
But by the time Steve Chu, now director of Lawrence Berkeley National Labs, arrived in Holmdel in 1983, the atom-trapping work "had been closed down for maybe three or four years," he says. Chu and Ashkin revived a small effort to trap large numbers of neutral atoms for fundamental physics experiments.
Trapping beads arose out of frustration, Ashkin explains. "We were having trouble with the atom trapping, so I decided to try simpler particles, like submicron dielectric particles," he says. In 1986 the team demonstrated that a single, tightly focused laser beam could trap a dielectric sphere. This single-beam configuration, known as optical tweezers, has since been used in thousands of papers. "It was a happy accident," Chu says.
Ashkin continued to explore the biological applications of optical tweezers. In addition to trapping bacteria, he demonstrated that individual organelles could be manipulated, by pulling on mitochondria in a giant amoeba.
Chu, meanwhile, moved to Stanford where in 1990, his group reported how to use optical tweezers to stretch individual molecules of DNA. He followed that work with a series of papers describing single-molecule studies of polymer dynamics using DNA as a model polymer. He also continued with atom-trapping experiments, for which he received a Nobel Prize in physics in 1997. Those techniques, in turn, formed the basis for the demonstration of Bose-Einstein condensation that garnered the physics Nobel in 2001.
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."
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.
How has the optical trap transformed the life sciences: Spawned a transition from population-based biochemistry to single-molecule biophysics
When was it developed: 1986
Primary application: Manipulating individual proteins in vitro
Pros: Illustrates the behavior of single molecules
Cons: Technically challenging and expensive
Key reference: A. Ashkin et al., "Observation of a single-beam gradient force optical trap for dielectric particles,"
Clinical application: None, yet
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
Invention of laser
Art Ashkin levitates micron-sized particles against gravity
Demonstration of single-beam, gradient-force optical trap, better known as optical tweezers
Ashkin manipulates viruses and live bacteria in the optical trap
Optical trap used to manipulate live sperm
Steven Block and Howard Berg measure stiffness of bacterial flagella
Steve Chu reports optical manipulation of DNA at the International Quantum Electronics Conference
Block uses trap to study movement of kinesin on microtubules
Michael Sheetz probes motion of glycoproteins in membranes
Karl Greulich and colleagues use optical tweezers to isolate individual chromosomes
J.J. Krol develops multiple-trap system
James Spudich, Chu, and Robert Simmons create a force trap with an active feedback circuit and use it to measure the force produced by a few myosin motors
Spudich (below) measures step size of myosin; Steve Block measures step size for kinesin
Jeff Gelles and Block measure force of an RNA polymerase molecule
Carlos Bustamante and Simmons separately measure the folding and unfolding of titin
Koen Visscher and Block develop molecular force clamp, which can maintain constant force on a single kinesin molecule
Block builds first combination optical trap/fluorescence instrument