The term “optical trapping” is perhaps misleading: It implies little more than grabbing a molecule or other object and possibly transporting it to some other place. But, as is apparent from studies that have emerged recently, optical trapping is a rapidly maturing technique whose utility extends far beyond what its name suggests.Carlos Bustamante at the University of California, Berkeley, is a researcher whose lab is devoted to the development of single-molecule methods such as optical trapping. The most significant technological advances of the past several years, he said, have been the development of high-resolution instruments and attempts to combine optical trapping with fluorescence detection and similar techniques. The former is especially exciting, he noted, because it allows investigators to follow single-molecule dynamics.Researchers have observed the folding and unfolding trajectories of individual RNA molecules by hybridizing a “DNA handle” onto one end of the growing RNA chain and using it to tug on the molecules with optical trapping. They first showed that they could alter the behavior of RNA polymerase (RNAP) using the method. In a more recent study, they described a DNA template with a roadblock on it. The RNA polymerase will transcribe a template and continue transcription until it encounters the roadblock. The piece of RNA hanging off the stalled polymerase complex is allowed to fold, and the researchers use the trapping technique to study this process.Traditional bulk methods typically provide an ensemble average of the details of a molecular cycle, and for this reason can offer only an idealized average picture of the molecular process of interest. Single-molecule methods such as optical trapping eliminate this ensemble average because they make it possible to follow the actual trajectories of the individual molecules. In particular, optical trapping now has high enough resolution that researchers can discern conformational changes at the angstrom level, for example. This makes it possible to follow molecular trajectories with unprecedented detail, Bustamante said. He outlined recent advances in the field in an Annual Review of Biochemistry paper published online on Feb. 28, 2008.Watching events unfoldResearchers with Stanford University in California, the University of Alberta, in Edmonton, Canada, and the National Research Council of Canada, also in Alberta, recently used optical trapping to observe the folding and unfolding trajectories of individual RNA molecules. As reported in the Feb. 1, 2008, issue of Science, they investigated the formation of riboswitches — elements that regulate gene expression by way of changes in mRNA structure — essentially by pulling on them and seeing what happened.Steven M. Block, the principal investigator of the study, described the assay, which was originally developed by his group. The researchers hybridize a DNA handle onto one end of the growing RNA chain as it emerges from RNA polymerase — the enzyme that synthesizes RNA. They attach a microscopic bead to the handle so they can exert forces with respect to the polymerase molecule by means of a pair of laser-based optical traps. The polymerase molecule is attached to a second small bead. “In other words, we can tug directly on the RNA,” he said.They showed in a previous paper that they could modulate the behavior of RNA polymerase using the method. In the study reported in Science, they developed a DNA template with a “roadblock” on it. RNA polymerase will transcribe a template and continue transcription until it runs into this roadblock. “As a result, we have a stalled polymerase complex with a piece of RNA hanging off it, which is allowed to fold,” Block said. Thus, they can use the technique to study folding and unfolding by applying force after the transcription process is completed. In this way, they monitor folding and unfolding by single-molecule force spectroscopy, measuring the extension of a molecule as it unfolds and refolds under external tension, thus enabling investigations of structural transitions.The researchers used a dual-trap device that split the beam from a 1064-nm Spectra-Physics laser into two separately controlled traps by polarization — one of which was much stiffer than the other — to facilitate the tugging of molecules. They steered the strong trap in the specimen plane using acousto-optic deflectors made by IntraAction Corp. of Bellwood, Ill. They monitored the position of the bead held in the weaker trap with subnanometer precision by observing the scattered light from an 830-nm laser from Point Source of Hamble, UK, using a duolateral position-sensitive detector from Pacific Silicon Sensors of Westlake Village, Calif.The real-time automated tracking and trapping system creates a region of interest centered on a sperm (the green box in this image represents the region of interest). By calculating the sperm’s swimming velocity and thus tracking the sperm, the system can then trap the sperm (the optical trap is represented by the smaller red box). Having done so, it reduces the laser power until the sperm can escape the trap and in this way determines the escape power — helping to confirm hypotheses about which species’ sperm swim faster and more strongly.The experiments yielded intriguing findings. First, the investigators found that the system folds through five distinct structural states as it collapses. Also, they observed aspects of structural formation that they did not anticipate and that even ran counter to conventional wisdom. In the area of nucleic acid folding, Block said, researchers have long believed that all secondary structures form first, followed by formation of any tertiary structures. The investigators noted, though, that the formation of secondary and tertiary structures is, in fact interleaved for this particular riboswitch, adhering to the following order: secondary, secondary, tertiary, secondary. “This violates the simpler paradigm of all secondary structures first forming, and only then tertiary structures forming,” Block said.The study was important because it yielded enough information to assign all five of the structural states of the folding process, to determine which elements are forming at what time and with what energies. It was also important, Block said, because “it showed we can do this at all!”Gaining momentumThe assay could help to answer a number of questions about gene expression. Transcriptional regulation is by far the most common form of gene regulation, Block said, and the levels of genes that are transcribed are often modified by pauses and other signals, such as terminators, that are encoded by sequences found in the DNA or RNA. Block’s lab has long been concerned with how these signals regulate behavior and will continue to study these using the new assay.“People are doing experiments now that were really just pipe dreams a few years ago,” Block said. “The notion that you could even see a single molecule is relatively new. Now you can study motor proteins, folding problems, assembly and ligand binding. It’s a terrific time to be doing this kind of biology.”The other notable development in optical trapping, Bustamante explained, is how widespread it has become. “The pace has accelerated significantly,” he said. “At meetings, I’m sometimes surprised at how many sessions and how many posters there are.” Much of this increase can be attributed to people moving into the field, which itself is a testament to how robust the field has become. “There are enough people working in this area, enough publications and results, that other people are getting excited about it,” he said. And the learning curve has gotten better, he added. With so many more publications and labs working in the area, opportunities to learn about the technique have risen greatly.In other cases, of course, increased application has been the result of technological innovation. For example, in the March 6, 2008, issue of the Journal of the Royal Society Interface researchers at the San Diego, Davis and Irvine campuses of the University of California, as well as at Omaha’s Henry Doorly Zoo in Nebraska, reported that they had combined optical trapping with computer-tracking software and robotics to study sperm competition and motility in primates.Although they had already demonstrated use of optical trapping to study sperm interactions and motility, the previous study had suffered from low throughput: They needed to trap the sperm manually and perform the various analyses off-line. Incorporation of the computer-tracking software and robotics helped them increase the throughput significantly.The researchers used the system to test an evolutionary theory about sperm competition in primates: namely, that species with multipartner matings (in which sperm from several males compete to get to the egg in the female) have evolved sperm that swim faster and more strongly than those found in monogamous species such as the gorilla.Principal investigator Michael W. Berns was trained in evolutionary biology but has spent much of the past several decades developing and using laser technology to study cells and tissue. He came up with the idea to use optical trapping to study sperm competition after attending a presentation on sperm competition that was given at the San Diego Zoo Center for Research on Endangered Species; he reasoned that measurements of sperm swimming force would complement more commonly used parameters, such as swimming velocity and lateral head movement in studies of sperm motility.“It occurred to me that we could use laser tweezers (traps) to hold the sperm,” he said, “and gradually reduce the trap until the sperm could swim out. This should then give us a quantitative measurement for the force that the sperm was swimming with.”In the present study, the investigators, led by Berns’ electrical engineering graduate student Jaclyn M. Nascimento, investigated the force and speed of sperm swimming and the relationship between the two parameters for the chimpanzee, rhesus macaque, human and gorilla. These primates represent a range of mating patterns. Gorillas exhibit one male/multiple female arrangements and, therefore, from the female perspective are monogamous. Chimpanzees and rhesus macaques both have multiple male/multiple female mating systems. Humans, across cultures, show a range of behaviors: one male/multiple females in 83 percent of societies, strictly monogamous in 16 percent of them and polyandrous — that is, with multiple male partners — in less than one percent.The primary disadvantage to using optical trapping for such an investiga-tion is that each sperm must be measured individually. This is why the researchers developed the sophisticated tracking software and robotic control of the microscope and optical trap used in their study. “We had to [develop] all the software and hardware for pattern recognition as the sperm swims,” Berns said, “and then [for] the physical control of the microscope so it could become relatively automated.”Although one of the scientists still had to select the sperm with the computer mouse to initiate the program, the advances helped to increase the number of sperm observed to between 100 and 200 in a two hour sitting, which was more than sufficient for their purposes. “Automated cell sorters look at millions of cells in that time frame,” Berns noted, “but our needs dictated individual sperm analysis.”The researchers performed the measurements using an optical trap generated by a 1064-nm Spectra-Physics laser coupled to a Zeiss microscope with a 40×, 1.3-NA oil-immersion lens. Video-rate phase contrast images of swimming sperm were digitized, and the real-time automated tracking and trapping system (RATTS) created a region of interest centered on the targeted sperm. The system calculated the sperm’s swimming velocity based on the pixel coordinates of its trajectory.RATTS established a stable average velocity after 3.33 s, or 100 frames, and then trapped the sperm. It reduced the laser power and determined when the sperm could escape the trap, recording laser power at the moment of escape. This measure was then converted to escape force.The experiments confirmed the initial hypothesis that sperm from species with multipartner matings swim faster and with more force than those found in species with single-partner matings. At the same time, they showed the feasibility of using optical trapping with the novel software and robotics system for investigations of sperm interactions and sperm motility.Moving beyond the “video game”The next step for optical trapping may be the commercialization of analytical instruments. “There aren’t really any analytical instruments on the market,” Bustamante said. “There are mostly just qualitative types of instruments where you trap objects and move them from one place to another — a little bit like a video game, where you grab cells and move them. Of course, that’s not really where the science is.”The important thing, he added, will be to develop commercial instruments that resemble the ones currently in laboratories. What are needed are not just qualitative instruments that allow someone to apply forces to microscopic objects but analytical devices that tell exactly how much force is being applied; instruments, he noted, that also can measure the forces developed in the course of chemical reactions. The key, though, is that these instruments must be user-friendly so that all researchers can use them, not just initiated physicists.Development of truly robust combined systems — with fluorescence detection, for instance — also would help to spur commercialization. Such systems would allow researchers to follow various chemical reactions or biochemical processes investigators simultaneously. As an example, investigators could follow a molecule moving along a track while monitoring changes inside the molecule. Such abilities would help to advance optical trapping still further.