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Surgical Applications of Femtosecond Lasers J. Biophoton. 2, No. 10, 557–572 (2009) / DOI 10.1002/jbio.200910053 Journal of BIOPHOTONICS REVIEW ARTICLE Surgical applications of femtosecond lasers Samuel H. Chung*; 1 and Eric Mazur2 1 School of Engineering and Applied Sciences, Harvard University, 9 Oxford Street, Cambridge, MA 02138, USA 2 Department of Physics and School of Engineering and Applied Sciences, Harvard University, 9 Oxford Street, Cambridge, MA 02138, USA Received 20 December 2008, revised 19 June 2009, accepted 27 June 2009 Published online 21 July 2009 Key words: subcellular laser ablation, nanosurgery, femtosecond lasers PACS: 06.60.Jn, 42.66.–p, 87.16.–b, 87.19.L– Femtosecond laser ablation permits non-invasive sur- geries in the bulk of a sample with submicrometer reso- lution. We briefly review the history of optical surgery techniques and the experimental background of femtose- cond laser ablation. Next, we present several clinical ap- plications, including dental surgery and eye surgery. We then summarize research applications, encompassing cell and tissue studies, research on C. elegans, and studies in zebrafish. We conclude by discussing future trends of femtosecond laser systems and some possible application directions. C. elegans dendritic bundle and femtosecond laser abla- 2 µm tion of middle dendrite (reproduced from [123]). # 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1. Introduction Femtosecond laser ablation has been applied to a wide range of research in materials science and the In the search for new methods of creating finer dis- life sciences. Some of the first researchers transi- sections with minimal collateral damage, femtose- tioned from micromachining transparent materials to cond laser ablation is emerging as an exquisite tool to ablating living cells and tissues. Others interested in perform non-invasive, submicrometer-sized surgeries laser tweezer manipulation or multiphoton micro- in the sample bulk. In addition, advances in laser scopy discovered they could permanently disrupt technology are making femtosecond laser systems their targets by increasing the laser power. Physicists more accessible in terms of cost and maintenance. As concerned with a theoretical understanding of laser a result, the number of research laboratories and clin- ablation proceeded to model the effects of shorter ical settings using femtosecond laser ablation has in- pulses as they became readily available. Biologists creased significantly in recent years, leading to a ra- and clinicians interested in a more precise tool for pid growth in the number of publications in the field. their applications have also joined the field. Because * Corresponding author: e-mail: [email protected] # 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Journal of BIOPHOTONICS 558 S. H. Chung and E. Mazur: Femtosecond laser surgery the femtosecond laser ablation field is so diverse and absorption. The remainder of this review paper fo- interdisciplinary, the literature contains many synon- cuses on applications of this last method, which can ymous terms for surgery by femtosecond lasers, de- yield higher resolution within the sample bulk with- pending on the application. A sampling includes out relying on labels. Reviews of the theoretical and laser microbeams, femtosecond laser surgery, subcel- experimental background of pulsed laser ablation lular laser ablation, nanosurgery, nanoscissors, nano- are available in the literature and range from the processing, nanoscalpels, nanodissection, nanoneuro- presentation of a simplified model (e.g., Section 2 of surgery, and nanoaxotomy. For simplicity, we will use [23]) to in-depth examinations of the ablation me- the general term femtosecond laser ablation through- chanisms [24–26]. out. Also, pulsed lasers have numerous applications, Nonlinear absorption occurs when the incident including laser tweezers [1], optical coherence tomo- optical intensity is sufficient to stimulate the simulta- graphy [2], stimulation of neurons [3], catapulting neous absorption of multiple photons. To minimize [4], and even artwork restoration [5]. Many of these the pulse energy and collateral damage, this is typi- techniques are worthy of a review in their own right, cally done using pulsed lasers with pulse durations but in this review we shall focus on the history, ex- of nanoseconds or less. Nanosecond and picosecond perimental background, applications, and future laser ablation has become a mainstay in many biol- trends of high-precision surgeries by femtosecond la- ogy laboratories and industrial applications. Im- ser ablation. provements in generating and amplifying femtose- cond lasers in the early 1980s made ultrashort laser pulses available in the laboratory, reducing the pulse energy needed for surgery and improving resolution 2. Historical background [27, 28]. The first studies employing femtosecond la- ser pulses for surgery were published in 1987–91 Surgery by electromagnetic radiation was first de- and examined laser interaction with retinal tissue monstrated in 1912 using an ultraviolet (UV) mi- [29, 30] and skin [31]. crobeam [6]. The introduction of the laser in 1960 [7] made high collimated intensities readily available in the laboratory, and surgical applications rapidly followed [8, 9]. A number of conventional (i.e., non- 3. Experimental background femtosecond) lasers have been used to perform dis- sections on a variety of biological media, including A number of papers have reported on the efficacy the retina [10], cellular organelles such as mitochron- and precision of femtosecond laser ablation, and dria [11, 12], the algae Spirogyra [13], skin cells [14], others have detailed the post-surgery viability of the mouse and rat melanoma [15], teeth [16], cell mem- cells, tissues, and organisms. Ablation was confirmed branes for micropuncture [17], chromosomes [18], to depend strongly on pulse duration [32], numerical and chloroplasts [19]. aperture (NA), and pulse energy [33]. Ablated areas While the transparency of many biological media were probed by scanning electron microscopy allows for direct optical observation of phenomena, (SEM) [34], atomic force microscopy (AFM) [35, it can also prevent absorption of laser light, which is 36], and fluorescence microscopy to ascertain the necessary for surgery. There are three methods of completion and resolution of surgery. Studies of fem- overcoming this obstacle: first, the target of interest tosecond laser ablation in a new medium typically can be labeled with a chromophore or dye that ab- establish the fundamental surgery characteristics, sorbs at the laser wavelength. The laser energy is de- such as surgical resolution and ablation threshold posited as destructive thermal energy [20] or the la- (usually for a fixed pulse duration and NA). bel breaks down, releasing free radicals which In living samples, post-surgery examination of destroy nearby chemical bonds [21]. While this tech- viability is essential. A few of the first studies using nique has a high resolution, labeling a single struc- femtosecond lasers observed the decreased cloning ture with an exogenous chemical can be difficult, efficiency of cells restrained by optical traps [37–39] and labels can be toxic to the sample. Second, one or imaged with two-photon microscopes [40]. Some can use a laser supplying a short enough wavelength cells become giant cells with multiple nuclei [41], so that linear absorption occurs. An example is UV and others undergo apoptosis because of laser-gener- “microscissors”, which have found wide use in ated reactive oxygen species [42]. However, proteins whole-cell ablations. One drawback is that linear ab- involved in recognizing and repairing DNA are also sorption leads to deposition of laser energy and present after surgery to mitigate damage [43]. Cellu- varying amounts of damage all throughout the beam lar viabilities and survival rates after femtosecond la- path, not only in the targeted volume [22]. Third, ab- ser ablation are generally high, and many studies sorption at the laser wavelength can occur by mov- present data from controls to confirm confinement ing to short laser pulses and stimulating a nonlinear of surgical effects. # 2009 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.biophotonics-journal.org RREVIEWEVIEW AARTICLERTICLE J. Biophoton. 2, No. 10 (2009) 559 There are a number of practical considerations nail [47]) and otology (inner and middle ear [48]), when implementing femtosecond laser ablation for this research has not yet generated a critical mass of surgery. First, if the target structures are in the sam- interest, perhaps because other simpler laser systems ple bulk, the sample must be transparent at the laser or treatment methods exist. wavelength and the target volume must be within the working distance of the microscope objective. Any significant linear absorption results in absorp- tion and collateral damage throughout the laser 4.1 Dental surgery beam. Second, a major disadvantage of femtosecond laser systems is cost. Oscillators, which generate fem- Initial conventional laser ablation studies on teeth tosecond pulses, are pumped by another laser, add- using ruby or CO2 lasers noted significant thermal ing to cost. Amplified systems often used for surgery deposition leading to collateral tooth damage [16, are approximately double the cost of a commercial 49]. Other lasers, such as Er : YAG, reduce thermal oscillator system. Fortunately, some newer high- effects considerably but cannot compete with the power oscillators and fiber lasers
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