Nanosurgery is forever hitting the headlines as research groups around the world explore the myriad applications for this precise technique. Couple this to the development of lasers with ever shorter pulse durations, and it’s little wonder that nanosurgery is creating quite a buzz in laboratories the world over. Today, nanosurgery is employed mostly as an in vitro technique for cell and tissue manipulation and as an in vivo technique in model organisms. However, with future developments of hybrid technologies, such as pairing laser sources with imaging systems and developing safe and reliable biological methods for in vivo operation, nanosurgery is set to become a formidable method in medicine. Possible applications include gene therapy, nerve regeneration and cancer treatment involving the selective damage of tumoral cells. High-throughput in vivo femtosecond laser surgery allowed discovery of drug leads that enhance neuronal regeneration. The first two panels show dramatic enhancement of axon regeneration when subjected to two potent chemicals with respect to a control (far right) without any chemical. Courtesy of Yanik Lab at MIT. The first observations at the nanoscale were achieved nearly 80 years ago with the advent of electron microscopy in 1931. Manipulation at such a scale, however, had to wait another 25 years for the surgical removal of subcellular organelles (mitochondria) in the 1950s. The early 1960s saw the invention of the first lasers and, since then, optical manipulation at the nanoscale has become a key technology in many new fields of research. Some of the main application areas include nanomechanics in surface physics, nanoelectronics in quantum computation, nanofabrication in materials science, molecular dynamics in biology, cellular and subcellular manipulation, and tissue engineering, among others. A femtosecond laser severed the dendrite in this AFD neuron, which is the primary type of neuron underlying thermotactic behavior. Courtesy of Samuel Chung/ Mazur Group, Harvard University. Precise nanosurgery ensures that nothing other than the targeted structure is damaged within the cell or organism. This allows scientists to probe and study important subsequent biological processes. For example, in groundbreaking research currently under way at MIT in Cambridge by associate professor Mehmet Fatih Yanik and at the University of Texas at Austin by associate professor Adela Ben-Yakar, nanosurgery is being used to sever neural processes and to observe nerve regeneration. Shown is an actin filament retracting over time after ablation with a femtosecond laser. Courtesy of Iva Maxwell/Mazur Group, Harvard University. “Nanosurgery allows the ablation of structures of sizes as small as 300 nanometers like actin filaments, cytoskeleton, cisternae of endoplasmic reticuli for cellular organelles or axons, dendrites or synapses for neural processes,” said Ben-Yakar, who is also the director of FemtoLab (FEMTOsecond Laser-Assisted Biomedicine). “It goes without saying that collateral damage is not an option when surrounding tissue may actually provide a structural cue to regeneration.” Shown is a model of femtosecond laser nanosurgery- on-a-chip for nerve regeneration studies in vivo using a whole organism, C. elegans. The chip allows chemical-free immobilization of the worm for precise laser nanosurgery of its axons. The full automation of this chip now provides the possibility for high-throughput screening of genes affecting nerve regeneration. Courtesy of Ben-Yakar Group. Nanosurgery advances Progress in nanosurgery currently pursues two avenues of research: One path relies on mechanical and robotic tools with nanoscale cutting precision, while the other relies on optical methods, which include taking advantage of the unique properties of lasers and their interaction with plasmonic nanoparticles. “Researchers are investigating several scientific avenues to achieve mechanical and robotic tools for nanosurgery, from plasma jets to nanorobots,” said Ben-Yakar. “Each method offers specific applicability, depending on which organs are being operated on.” A femtosecond laser cut these microtubules, causing their depolymerization in mitotic spindle (left). Courtesy of Valeria Nuzzo/Mazur Group, Harvard University. Advances in ultrashort pulsed laser technology provide an attractive alternative to chemical and mechanical approaches for cell and tissue manipulation. Indeed, femtosecond laser ablation, in addition to being a noninvasive and reliable technique, can be used to perform very accurate and selective surgery, as the nonlinear laser-material interaction is confined to the focal volume. For instance, a cell organelle can be dissected while leaving the cell membrane intact. What’s more, femtosecond irradiation ensures nanoscale precision as well as reduced mechanical and thermal effects. Eric Mazur, Harvard University physics professor, discusses developments in ultrafast lasers. Courtesy of Stephanie Mitchell/Harvard University News Office ©2002. “The last two decades have witnessed strong advances in laser technology for decreasing pulse duration and increasing pulse energy,” said Eric Mazur, professor of applied physics at Harvard University in Cambridge, Mass. “Among them are the development of the titanium:sapphire crystal and rare-earth-doped glass as lasing materials for the generation of very short pulses, and the introduction of the chirped pulse amplification technique for safely increasing the pulse energy. “These developments have made ultrafast lasers more accessible in terms of cost and maintenance, enabling short-pulsed lasers to be used in research as well as in clinical settings. In parallel with laser advancements, on the biological side, the cloning of the green fluorescent protein has been extremely useful in localizing particles targeted by the laser,” he added. A clinical endoscope is used to perform image-guided femtosecond laser microsurgery of cancer cells. The inset shows two-photon fluorescence images of cancer cells taken with the prototype (left image) and the same region after the targeted ablation of a single cell. The targeted cell was instantly destroyed, while neighboring cells were left intact. Courtesy of Ben-Yakar Group, University of Texas, Austin. Nerve regeneration According to Yanik, who heads up the High-Throughput Neurotechnology Group at MIT, some of the biggest breakthroughs have been in the innovative applications of nanosurgery for various investigations that would otherwise be impossible. “Since most cellular processes vary on nanometer-length scales, optical manipulation at nanoscale resolution opens up a new dimension in cellular and subcellular scale investigations,” he said. Working together in 2004, for instance, Yanik and Ben-Yakar used nanosurgery to demonstrate the first study of neuronal regeneration in a small multicellular organism (Caenorhabditis elegans). Several biology labs have subsequently adapted the technique, and in the last C. elegans meeting, there were several novel findings made possible by nanosurgery. High-throughput microfluidics captured and immobilized the C. elegans seen in this screening chip. Neurons that are labeled by a green fluorescent protein reporter are visible inside the optically transparent worm. Courtesy of Yanik Lab at MIT. In 2007, Yanik’s group developed a high-throughput microfluidic chip technology to manipulate C. elegans. Subsequently, in 2008, teams under Yanik and Ben-Yakar independently demonstrated a microchip that can rapidly perform nanosurgery on C. elegans. Yanik’s technology also allowed high-throughput in vivo neuronal regeneration screens using large drug libraries and genetic factors affecting neuronal regeneration. Since then, the Yanik group has discovered highly potent drug leads that enhance neuronal regeneration. The lab is now testing these on human neurons derived from stem cells. More recently, the Ben-Yakar Lab developed a fully automated microfluidics chip that can perform femtosecond laser nanosurgery of axons without human intervention. This new automated platform now allows genome-wide screening of nerve regeneration in a high-throughput manner. “This microfluidic device allows us to precisely manipulate C. elegans inside small channels and eliminates the need for anesthetics, which have shown to interfere with the axonal regrowth process,” Ben-Yakar said. “With tools such as laser nanosurgery and microfluidic devices and their full automated integration, we can undertake gene and drug screenings that usually need high-throughput systems because of the sheer amount of samples involved. Whole-genome screening is now conceivably possible within two years.” Cancer treatment The ability to selectively and precisely ablate biological tissue is a natural candidate for the treatment of cancer. In this field, nanoparticle-assisted surgery activated by lasers shows great potential. Nanoparticles conjugated with specific molecules (for example, ligands or antibodies) can anchor onto specific cells or tissue. Once exposed to a laser beam, the nanoparticles act as lenses and heat and/or ablate the targets they are attached to, such as cellular membranes or mitochondria. “Plasmonic laser nanosurgery is a novel photodisruption technique that exploits the large enhancement of femtosecond laser pulses in the near-field of metal nanoparticles for the selective and nonthermal nanoscale manipulation of biological structures,” Ben-Yakar said. “We have demonstrated the feasibility of plasmonic laser nanosurgery for both cellular death by necrosis for cancer treatment and transient pore formation for cellular transfection.” Precise imaging needed According to Yanik, a promising line of research that is yet to be explored is the use of coherent ultrafast pulse-shaping techniques to increase precision, chemical selectivity and repeatability of nanosurgery. But perhaps the most important work needs to be done in the field of microscopy. “Nanosurgery is a blind operation without superresolution microscopy,” Yanik said. “Thus, I believe there is significant need to combine nanosurgery with new microscopy techniques such as STED (stimulated emission depletion microscopy), stochastic optical reconstruction microscopy/photoactivated localization microscopy and structured imaging.” In fact, the MIT group recently proposed the use of electron beams for noninvasive molecular-scale imaging, which it hopes may one day be used to simultaneously image and perform molecular-scale surgery with electron beams. Nanosurgery research may still be in its infancy, but with so many exciting breakthroughs in the field, there can be little doubt that this mighty tool is coming to a surgery table near you.