Targeting single cells with a laser microscalpel
Femtosecond laser microsurgery (FLMS), which employs ~10−13s laser pulses, precisely destroys cells and smaller structures in 3D biological tissue.1–4 The technique uses high peak intensity pulses to create multiphoton ionization, thereby vaporizing targeted tissue. The process allows for the use of deep-penetrating near-infrared light and requires two to three orders of magnitude less energy than nanosecond pulses.3 This combination of high precision and tissue penetration makes FLMS appealing for delicate procedures and those requiring vaporization of diseased cells just beneath the surface. Since 2003 femtosecond lasers have been used clinically, specifically for LASIK surgery.5 Developing the technology for internal surgical procedures could greatly expand its reach.
Unfortunately, delivering the high peak intensity pulses through optical fiber and miniature optics has proven difficult. Furthermore, to fully exploit the precision of this technique, the microsurgery laser must be guided by equally precise and penetrating imaging. These challenges have prevented the integration of FLMS into an endoscopic surgical tool. Towards this goal, we have developed a miniaturized microscope which provides both imaging and femtosecond laser pulses, allowing the user to directly monitor the area of microsurgery.6
The miniaturized microscope, shown in Figure 1(a), uses a hollow-core photonic crystal fiber to guide the pulses through air. This approach eliminates the problems of material damage and the nonlinear effects that occur with conventional optical fiber. In addition to the pulses used for microsurgery, we deliver lower-intensity ones for imaging via the same optical pathway. This nonlinear technique, called multiphoton excitation, provides the same resolution and depth penetration as FLMS. By using similar pulses and identical optical pathways, the probe can precisely visualize the exact area being targeted.
Figure 1(a) shows the two-axis microelectromechanical systems (MEMS) scanning mirror, developed by our collaborator Olav Solgaard at Stanford Univeristy.7 It sits inside the probe housing and scans the laser pulses through a miniature lens pair. The pair images the mirror to the back aperture of a gradient-index objective lens. This allows uniform illumination of a wide field of view (FOV) across the mirror's entire scanning range. The lens pair also serves as a beam expander, enabling the imaging and microsurgery laser beams to overfill the back aperture of the objective lens and maximize focusing power.
The system was designed to provide subcellular resolution over a wide FOV and a fast frame rate, both crucial elements for guidance and navigation in vivo. The probe provides two-photon imaging at 10 frames per second, and has a maximum FOV of 310μm and a spatial resolution of 1:6μm. A dedicated collection fiber transmits the fluorescent imaging signal to a photomultiplier tube for image reconstruction.
We have demonstrated the combined imaging and microsurgery capabilities of the probe using breast cancer cells—cultured both in a monolayer and in a 3D collagen-based tissue phantom. In these experiments, we labeled cancer cells with calcein AM, a fluorescent dye that only activates in live cells. We performed microsurgery using one femtosecond pulse of 280nJ (~14 TW/cm2 peak intensity), detecting membrane disruption by a sudden loss of fluorescence in a single, targeted cell (see Figure 2). The use of one pulse and a spot size much smaller than the cell diameter eliminated the risk of signal loss due to photobleaching. We also showed the technique's precision in a 3D tissue phantom. In both demonstrations, the cells next to the targeted one remained intact. For clinical applications, the microsurgery laser could be scanned to quickly, and selectively, ablate cells in larger regions of interest.
This probe may allow clinicians to use a microscale, seek-and-treat tool for operating on otherwise inaccessible parts of the body. Our next steps aim to use alternative objective lenses to improve the resolution of the system. We are also miniaturizing the device's overall dimensions to make it small enough for endoscopy.
The authors would like to thank Wibool Piyawattanametha, Hyejun Ra, and Olav Solgaard for development of the MEMS scanning mirror and Nicholas Durr for technical assistance. This work was supported by National Science Foundation grants BES-0548673 and BES- 0508266, and National Institutes of Health grant R03-CA125774.
Adela Ben-Yakar is an assistant professor. The Fulbright scholar earned a PhD in mechanical engineering from Stanford University in 2000. From 2000 to 2004, she was a postdoctoral researcher in applied physics at Stanford and a visiting scholar at Harvard University. Her research interests are in femtosecond laser nano- and microsurgery, plasmonic nanoparticles for laser nanosurgery, and two-photon luminescence imaging. She also studies miniaturized laser microsurgery probes for diagnosis and treatment of cancer, and microfluidics for in vivo nerve-regeneration studies.
Christopher Hoy is pursuing a PhD in mechanical engineering at UT Austin. He earned a bachelor's degree from Virginia Tech in 2004 and a masters from UT Austin in 2007, both in mechanical engineering. His research focuses on optical instrumentation design for biomedical applications.
