Femtosecond lasers prepare to break out of the laboratory

Femtosecond lasers (FSLs) have always been a promising technology, but to date they have been largely restricted to use in cutting-edge university and government laboratory research. However, FSLs may finally be ready to break out of the laboratory and head into the marketplace, with such potential applications as tattoo removal, eye and dental surgery, arterial plaque removal, and destruction of viruses, bacteria, or even cancer cells.

Conventional continuous-wave (CW) laser ablation relies on linear absorption of light, in which the photon wavelength matches a molecular energy resonance in the material. “This process is fundamentally thermal since the molecules vibrate like a mass on a spring, heat up, then melt, combust, or boil off,” says Adam Tanous, director of applications marketing for Raydiance Inc, a manufacturer of FSL systems based in Petaluma, California. Thus CW lasers often create thermal damage, which limits their usefulness in many potential applications.

In contrast, FSL ablation relies on photoionization, concentrating a great deal of power into an extremely short time frame: mere femtoseconds. FSLs strip electrons from their bound positions in the atoms of target materials to create a localized plasma. “Without electrons, the local material lattice structure gains a positive charge and becomes unstable,” says Tanous. Because of the Coulomb repulsion between the positively charged centers of the target atoms, material is ejected, or vaporized, from the localized area (ablation). The process of plasma formation and ablation occurs much faster than the excess energy can be transferred in the form of heat to the surrounding material. Because very little heat is generated, there is little chance for thermal damage. Ultimately, the charged particles recombine.

FSLs have proven very useful in the laboratory, in part because of their ability to cut or drill material precisely without burning. For example, in 2003 Eric Mazur of Harvard University and his colleagues used FSLs to sever parts of biological cells’ internal protein skeletons and to cut a nerve cell’s connection without killing it. Scientists at Lawrence Berkeley National Laboratory have used FSLs in combination with spectroscopy to determine the chemical composition of solids. FSLs can also be used to create periodic nanostructures and build layers of such diverse materials as dielectrics, semiconductors, metals, plastics, and resins.

A versatile light

One emerging practical application for FSLs is ophthalmology, specifically LASIK surgery and corneal repair. IntraLase Corp (Irvine, California) offers FSL systems for refractive and corneal surgery, in which FSLs replace the microkeratome mechanical knife that makes the initial cut in the cornea. That company merged with Advanced Medical Optics Inc in March and is now adapting its FSL technology for corneal transplant surgery.

In dentistry, FSLs may one day offer a viable alternative to mechanical drills and CW lasers for removing the dental composite materials used in fillings without damaging the surrounding tooth enamel. Another potential application is removing plaque from clogged arteries, using FSL treatments instead of balloon angioplasty and the insertion of stents. The buildup of plaque causes arteries to harden, restricting blood flow. It may be possible to weave an optical fiber about the size of a human hair up through a tiny incision in the femoral artery and use an FSL to precisely ablate plaque from the arterial wall, cell by cell; that procedure would increase blood flow without damaging the arterial structure.

To date, however, FSL systems have been large, expensive, and difficult to manufacture in commercial-scale volume; they’re also not user-friendly and require highly trained specialists to operate them. Those constraints have severely limited the viability of FSLs for broader applications. Fresh interest is being fueled in part by a new miniaturized platform developed by Raydiance.

The company says it has combined advanced fiber- and micro-optical components from the telecommunications industry with the latest microelectronics technology. According to Raydiance president Scott Davison, the new platform gives researchers access to FSL systems that are not only powerful and versatile but also portable and easy to use. As with full-sized FSLs, the Raydiance technology can instantly vaporize material very precisely, without heat or residual damage, down to a resolution of just a few microns.

The difference is its smaller size—roughly the size of a slide projector—combined with proprietary software that enables quick and easy tailoring of the system’s parameters to meet specific needs, according to Davison. The Raydiance system also uses erbium-doped fiber-optic cables, as opposed to titanium sapphire, which is found in most full-sized FSLs. Although titanium sapphire FSLs generate the shortest pulses with the highest energy per pulse, they are also more expensive and cumbersome to use.

The next step, says Davison, is putting the system into the hands of scientists to test its full capabilities. Last summer the company entered into a two-year cooperative R&D agreement with the US Food and Drug Administration (FDA), which is currently testing the safety and effectiveness of the Raydiance platform for various biomedical applications. A Silicon Valley startup company called EpiRay is using the Raydiance system to develop an FSL technology to remove tattoos by ablating the dyes used to create the tattoo without burning the surrounding skin.

Researchers at the University of New Mexico are using the Raydiance platform as a source to perform laser-induced breakdown spectroscopy. In that technique, lower energies are employed to ablate a minute amount of the substrate. The ensuing plasma plume can then be used to generate the emission spectra of the atomic species present in the sample, says Tanous.

One day treating cancer with FSLs may even be possible. FDA researcher Ronald Waynant has been using the Raydiance system to study FSLs’ effects against cancer cells. His initial results indicate that FSL treatments can significantly slow tumor growth without the severe side effects associated with conventional chemotherapy and radiation therapy. Because FSLs are so precise, he believes it may be possible to erase cancer one tumor cell at a time.

Much of the research is still in the preliminary, proof-of-principle stage as researchers continue to test the new system’s capabilities. The tricky part in many biomedical applications in particular is determining the appropriate ablation frequencies of different forms of matter. Pinpointing those critical frequencies can be difficult for biological materials such as arterial walls or cancer cells.

Harvesting skin

In mid-November, Raydiance announced its most ambitious project yet: a collaboration with Rutgers University and the Musculoskeletal Transplant Foundation to use the company’s FSL platform to process donated tissue for skin transplants. The idea is that the FSL could be used to separate skin layers with minimal damage, to decontaminate tissue, and to speed up the process of tissue cleaning. MTF is the largest nonprofit consortium of academic medical institutions and organ and tissue recovery organizations in the US.

Skin transplants are critical for treating burn victims, who are especially susceptible to infection and fluid loss. They are usually performed on burn victims to protect the open wounds during healing, and they generally use the patient’s own skin, when possible, to avoid problems with organ rejection. More than 900 000 Americans receive tissue transplants every year, according to MTF president and CEO Bruce Stroever. However, harvesting enough usable skin to meet the growing need is difficult. Cadaver and animal skin have been used as temporary cover, but they are rejected fairly quickly. Immunosuppressants cannot be used because patients need their immune system to fight off infection.

The principal investigator at Rutgers, Zhixiong Guo, deems the power and precision of FSLs particularly promising for tissue engineering for skin transplants because it can maximize the amount of usable material processed from donated skin tissue. In principle, at least, the laser should be able to separate skin layers more precisely and effectively, while also decontaminating the surface of soft tissue and reducing the risk of infection.

The collaboration is the first test of its kind, and if successful, it will result in more usable tissue for those in need of transplants. Initially, the project will focus on proof of principle: developing noninvasive laser-ablation methods for separating the dermal and epidermal layers of the skin to provide improved viability of the limited supply of donor tissues.

A second focus in the initial stage will be to develop noninvasive sterilization techniques for donor skin and tendons to minimize damage to the tissue while removing viral and bacterial contamination. Finally, the researchers hope to develop a method that can effectively remove unwanted hair from donor tissue with minimal damage.

Killing viruses and bacteria

Kong-Thon Tsen, a physics professor at Arizona State University, has demonstrated that full-sized infrared FSLs can destroy common viruses and bacteria without harming surrounding healthy human cells. While strolling in the park over Christmas break in 2006, he and his son, Shaw-Wei Tsen—a student in pathology at the Johns Hopkins University—hit on the idea of applying the elder Tsen’s work on FSLs to develop antiviral, antibacterial treatments beyond conventional vaccinations that can lead to problems with drug resistance and clinical side effects.

Ultraviolet irradiation is an alternative technique for disinfection, but having no selectivity, it kills both the unwanted microorganisms and mammalian cells. One promising experimental technique to circumvent those limitations is microwave absorption. However, water usually coexists with the unwanted microorganisms and greatly absorbs microwave energy in the range of 10–300 GHz. That happens to be the typical vibrational frequency range of the capsids (protective protein shells) of viruses. Thus it is extremely difficult to transfer microwave excitation energy to the vibrational energy of viruses—and similarly to bacteria—without heating up the surrounding water molecules.

Tsen’s method targets the capsids and can inactivate the unwanted viruses while leaving sensitive materials like mammalian cells unharmed. The FSL coherently excites vibrations with large amplitude on the capsids through the impulsive stimulated Raman scattering process.

By tuning the FSL to just the right pulse or spectral width, combined with high enough intensity, the user can create resonant vibrations strong enough to break the weak links on the capsids; the damage to the capsids ultimately causes it to disintegrate. Tsen likens the effect to giving a child a push on a swing. “If the pushing force is constant, then the maximum amplitude is achieved when the force is applied for one-quarter of a cycle of the swing,” he explains. “Thus a window exists for pulse width that can be used for selectivity.”

The difference in size between viruses, bacteria, and mammalian cells is another critical factor. A virus typically measures 0.01 micron, compared with 0.1 micron for bacteria and 1 micron for mammalian cells. Because of the corresponding difference in surface area, more water molecules surround the mammalian cells than surround viruses or bacteria. The result is a dampening effect that minimizes the strength of the vibrations of the cells much more than it does for viruses or bacteria. According to Tsen, this offers a partial explanation of why it takes much larger laser intensity to damage cells.

To date, Tsen has successfully demonstrated the technique on the tobacco mosaic virus, the M13 virus, and the Escherichia coli bacterium. Atomic force microscopy images of irradiated M13 viruses indicate that his technique does indeed destroy viruses by damaging the capsids. However, bacteria have a very different structure—lipid bilayers rather than capsids—and Tsen has yet to perform AFM imaging on the laser-irradiated bacteria to determine whether the same process, disintegration of the protein coat, is responsible for their inactivation.

Much development work must be done to bring such a system into clinical use, but Tsen believes the first likely application of his technique might be to kill any virus and bacteria tainting the blood stored in blood banks. He is currently testing his technique on HIV and hepatitis viruses.

All in all, femtosecond lasers are shaping into an important enabling technology for promising breakthroughs in biology and medicine. Davison, for one, anticipates even more new applications in other market sectors as such systems continue to become smaller, cheaper, and easier to use. “It’s always been an incredibly versatile light,” he says of the FSL. “It’s just been trapped in big room-sized devices until now.”