LABORATORY SCIENCE

Characterization of submicrojoule femtosecond laser corneal tissue dissection

Perry S. Binder, MS, MD, Melvin Sarayba, MD, Teresa Ignacio, MD, Tibor Juhasz, PhD, Ronald Kurtz, MD

PURPOSE: To document the acute morphologic features of laser of situ keratomileusis (LASIK) flaps created using an IntraLase femtosecond laser (IntraLase, Inc.) with a 60 kHz engine. SETTING: Laser suite in a clinical practice. METHODS: A LASIK flap was created in 4 human eye-bank eyes using the 60 kHz IntraLase femtosecond laser with the following settings: 110 mm flap thickness, 9.0 mm flap diameter, 60-degree hinge length, 65-degree side cut, 0.4 mJ or 0.7 mJ raster energy, 7 mm 7 mm or 9 mm 9 mm spot/ line separation, and 1 mJ side-cut energy. Immediately after the laser pass and without the flap being lifted, the globes were placed in fixative and subsequently processed for light and transmission electron microscopy. RESULTS: All 4 procedures were completed without complications or the appearance of an opaque bubble layer. The flaps were of uniform thickness and equaled the attempted thickness. Some areas had a complete dissection; other areas had scattered, incomplete tissue bridges. The adjacent corneal stroma and keratocytes were uninjured. When the epithelium was removed, the stromal component of the flap was measured as the attempted thickness; when the epithelium was present, the total flap thickness approximated the attempted flap thickness. CONCLUSIONS: Laser in situ keratomileusis flaps were safely created using raster energies and laser spot separations below those being used clinically. This technique may allow creation of flaps that are reproducibly thinner than those currently being performed and thus confer the benefits of surface ablation and LASIK. J Cataract Refract Surg 2008; 34:146152 Q 2008 ASCRS and ESCRS

The mechanical microkeratome has been used to create the laser in situ keratomileusis (LASIK) flap since the procedure was introduced by Pallikaris et al.1 Although mechanical microkeratomes have continually improved in their construction and ability to safely

Accepted for publication July 30, 2007. From a private practice (Binder), San Diego, and IntraLase, Inc. (Binder, Sarayba, Ignacio, Juhasz, Kurtz) and the University of California (Juhasz, Kurtz), Irvine, California, USA. Sponsored by IntraLase Corp. Drs. Sayarba, Juhasz, and Ignacio are paid employees of AMO/ IntraLase, Inc. Drs. Binder and Kurtz are paid consultants to AMO/IntraLase, Inc. Corresponding author: Perry S. Binder, MS, MD, 8910 University Center Lane, Suite 800, San Diego, California 92103, USA. E-mail: garrett23@aol.com.

create LASIK flaps,2,3 the achieved flap dimensions are still dependent on blade quality, the gap width of the microkeratome head,4 preoperative corneal curvature and thickness,5 intraocular pressure (IOP), and translation speed.6 With most microkeratomes, the shape of the created flap is thicker at the entrance and exit sites of the blade and thinner in the center, the so-called meniscus-shaped flap.7 Because of the need to improve the accuracy of flap creation, surgeons turned to the use of laser technology to create LASIK flaps. As early as 1996, surgeons began attempting to create a corneal flap using picosecond lasers; unfortunately, the dimensions of the laser ablation were large and created significant corneal bridges, resulting in an unsatisfactory, rough dissection.8 In 2001, the IntraLase femtosecond laser (IntraLase Corp.) with an engine speed of 6 kHz was introduced in the United States; with 6 Hz, it took more than 2 minutes to create a 9.0 mm corneal flap.9 Subsequent increases in the frequency of the
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emission of pulses of the IntraLase engine have increased the speed to 60 kHz, which permits creation of a 9.0 mm flap in fewer than 20 seconds. Scanning electron microscopic studies of the interface created by the 15 kHz and 30 kHz engines,10 and more recently with the 60 kHz engine,11 report the ability to create stromal surfaces whose smoothness has been graded qualitatively and quantitatively to be equal or superior to surfaces created with mechanical microkeratomes. In addition, clinical studies comparing femtosecond laser LASIK outcomes with those of mechanical microkeratomes found IntraLasecreated flaps produce excellent visual and refractive outcomes1214 while minimizing induced higher-order aberrations (HOAs).15 The physics of the femtosecond lasergenerated microplasma ablation has been tested in vitro and in vivo.1619 The microplasma bubble expands from an initial 2 mm to 5 mm in diameter, creating bubbles several times larger in diameter. Adjacent plasma bubbles overlap so that a contiguous dissection plane can be created. The dimensions of the spot size created with the picosecond laser are well over 100 mm,17,20 whereas the initial dimensions of the femtosecond laser pulse are less than 5 mm.19 The newest 60 kHz IntraLase femtosecond laser is the most commonly used model. Although it creates predictable flap dimensions and permits surgeons to reduce the laser ablation parameters and energy settings, to our knowledge there have been no transmission electron microscopic (TEM) studies of a freshly created flap with this laser. The purpose of this current study was to study the immediate morphologic appearance of a femtosecond lasercreated corneal LASIK flap using light microscopy (LM) and TEM. MATERIALS AND METHODS Corneal Model
This study used human eye-bank eyes obtained between 6 hours and 24 hours postmortem. The IOP was increased by injecting balanced salt solution (BSS) through the optic nerve using a 27-gauge butterfly needle connected to a 5 cc syringe. The achieved IOP was tested by feeling the pressure inside the globe to be in a normal range (ie, not soft or hard). The surface of the globes was moistened by a stream of BSS,
Table 1. Laser settings used. Separation (mm) Specimen 1 2 3 4 Raster Energy per Pulse (mJ) 0.7 0.7 0.4 0.4 Spot 9 9 7 7 Line 9 9 7 7

with care not to use an excessive volume. The surfaces were gently dried with a cellulose sponge before the laser procedures to avoid creating a false applanation meniscus. If the epithelium was loose, it was gently wiped off Bowmans membrane. This technique has been used in previous morphology studies.10,11

Femtosecond Laser Settings

The attempted flap thickness was set to 110 mm, the flap diameter to 9.0 mm, and the hinge length to 60 degrees. Table 1 shows the other laser settings. The femtosecond lasers suction fixation ring was placed on each eye-bank eye and suction applied as is clinically performed under the operating microscope of the Visx excimer laser. The eye-bank eye affixed to the suction ring was then docked to the femtosecond lasers glass applanation cone. The surgeon observed the increasing diameter of the applanated cornea through the operating microscope of the femtosecond laser at a magnification of 6 until the entire area of the glass-applanated surface had a complete meniscus. The foot pedal of the laser was engaged until the entire raster pass was completed. Immediately after the side cut was completed, and without the flap being lifted, the globe was removed from the suction ring and placed in fixative. One to 2 minutes elapsed between the end of the raster pass and placement of the specimen in fixative. Subtraction ultrasonic pachymetry of the eye-bank eyes was not performed.

Light and Transmission Electron Microscopy

The sample was immersion fixed in glutaraldehyde 2.5% in 0.1 M Sorensen buffer, pH 7.4, at C4 C for several days. After several buffer rinses, the sample was postfixed for 1 hour in osmium tetroxide 1% in the same buffer. It was then rinsed in double distilled water to remove phosphate and then en bloc stained with aqueous uranyl acetate 3% for 1 hour. It was dehydrated in ascending concentrations of ethanol, treated with propylene oxide, and embedded in Epon epoxy resin. Semithin sections were stained with toluidine blue and basic fuchsin for LM. Selected regions of interest were ultrathin sectioned to 70 nm in thickness and stained with uranyl acetate and lead citrate. They were examined using a Philips CM100 electron microscope at 60 kV. Images were recorded digitally using a Hamamatsu ORCA-HR digital camera system operated with AMT software (Advanced Microscopy Techniques Corp.).

RESULTS Clinical Appearance The raster pattern in all 4 human eye-bank eyes was uniform and smooth. No eye had opaque bubble layer

Flap Diameter (mm) 9.0 9.0 9.0 9.0

Side-Cut Angle (Degrees) 90 60 90 60

Epithelium Intact Intact Removed Removed

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Figure 1. Specimen 1. IntraLase raster pass is 45% complete using a spot and line separation of 9 mm 9 mm and a raster energy of 0.7 mJ (700 nJ). The flap was not lifted before TEM preparation (original magnification 6).

formation. The procedures were completed in fewer than 24 seconds (Figure 1). Light Microscopy The planar nature of the LASIK flap was easily visualized over the dimensions of the created flap (Figures 2 to 5). In all 4 specimens, the stromal component of the flap was of uniform thickness. The measured flap

thickness was 5 to 10 mm thinner than the attempted 110 mm; tissue shrinkage artifact was the most likely explanation. When the epithelium was present (specimens 1 and 2), the stromal component of the flap appeared to equal the epithelial thickness (Figures 2 and 3); when the epithelium was removed (specimens 3 and 4), the stromal component of the flap equaled the attempted thickness; when the epithelium was present, the total flap thickness approximated the attempted flap thickness (Figures 4 and 5). This finding supports previous clinical studies of the accuracy of the software in planning the attempted flap thickness based on the distance from the bottom of the glass applanation plate. In some areas of each specimen, the contiguous nature of the bubble pattern was easily seen (Figure 3). In other areas in each specimen, partial or complete tissue bridges were present (Figures 2 and 4). There appeared to be more tissue bridges when the spot and line separations were 7 mm (specimens 3 and 4). The tissue bridges are not seen clinically when the surgeon lifts a flap created by the IntraLase laser. The diameter of the bubbles varied between 25 mm and 75 mm. At the LM level, there was no evidence of damage to the corneal stroma on either side of the interface or to the epithelium in the 2 specimens with an intact epithelium (Figures 2 and 3). In 1 specimen, gas-bubble creation was observed in the pocket that is used to allow the interface gas to escape adjacent to the hinge during the start of the raster pass (Figure 5). Just as in the interface, the surrounding stroma and keratocytes in the pocket area were intact morphologically.

Figure 2. Light microscopy of specimen 1 (original magnification 20); the flap was created using a spot/line separation of 9 mm 9 mm and raster energy of 0.7 mJ. The flap thickness appears uniform. There is an equal proportion of epithelial thickness and stromal thickness. The attempted flap thickness approximates the 110 mm attempted thickness (bar Z 100 mm).

Figure 3. Light microscopy of specimen 2 (original magnification 100). The flap thickness composed of 50% epithelium and 50% stroma approximates the planned flap thickness of 110 mm using raster energy of 0.7 mJ and a spot line separation of 9 mm x 9 mm. Some tissue bridges are seen inferiorly (bar Z 100 mm)

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Figure 4. Light microscopy of specimen 3 (original magnification 40) using a spot/line separation of 7 mm 7 mm raster energy of 0.4 mJ. The flap thickness approximates the 110 mm attempted as there was no epithelium in this specimen (Table 1) (bar Z 200 mm).

Figure 5. Light microscopy of specimen 4 (original magnification 100) using a spot/line separation of 7 mm 7 mm raster energy of 0.4 mJ. There was no epithelium in this specimen (Table 1). The flap thickness is uniform and approximates the 110 mm attempted. One tissue bridge is seen. The pocket dissection is seen in the superior aspect of the photograph, deeper than the start of the raster pattern as it is designed to allow egress of interface gasses (bar Z 100 mm).

Transmission Electron Microscopy On TEM, all specimens had smooth surfaces on both sides of the interface. Keratocytes immediately adjacent to the interface appeared undamaged (Figures 6 to 9). Some tissue bridges with an appearance like that of blunted stalactites and stalagmites were observed (Figures 7 and 8). Figure 6 shows a complete tissue bridge. There was no evidence of damage to the keratocytes above or below the interface or of morphologic disturbance to Bowmans layer or the epithelium. The tissue bridges on either side of the interface did not have a regular pattern. DISCUSSION Over 1 million LASIK flaps have been created with the IntraLase femtosecond laser (data on file, January 1, 2007, IntraLase Corp.). Clinical studies have shown that this technology creates flap dimensions that are more predictable than those produced with mechanical microkeratomes.13,2123 The achieved flap dimensions are independent of corneal diameter, thickness, and curvature.23 The clinical results of the laser-created flaps on the visual and refractive outcomes and induced HOAs are excellent.13,14 Although with the IntraLase laser surgeons are currently using raster energies in the range of 0.8 to 1.0 mJ (800 to 1000 nJ) with spot and line separations in the range of 8 mm 8 mm to 9 mm 9 mm, this study shows that surgeons can use lower raster energies and smaller spot line separations. Additional eye-bankeye studies and subsequent clinical studies will be necessary to confirm our findings. The physics of the femtosecond spot creation dictate that as one decreases the attempted spot size dimensions, one is able to decrease the energy required to create the individual spots.1619 Whereas with the IntraLase most surgeons use a spot/line separation of 9 mm 9 mm, we created equivalent flaps using a spot/line separation as low as 7 mm 7 mm. As one decreases the spot/line separation, one is able to decrease the raster energy to achieve the same effect. One of the benefits of decreasing energy is to decrease the potential for transient light-sensitivity syndrome.24 Decreasing the spot and line dimensions makes the flap dissection mechanically easier (personal observations since September 2005) while improving the smoothness of the interface, as seen under the operating microscope.10,11 However, each laser may have

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Figure 6. Transmission electron microscopy of the interface near the termination of the raster pattern in specimen 1. Both sides of the interface are smooth. A tissue bridge of approximately 8 mm is seen in the middle of the figure. There is no lamellar damage adjacent to the interface. The raster energy was 0.7 mJ and the spot/line separation, 9 mm 9 mm (bar Z 2 mm).

minor variations in the laser output; therefore, similar laser settings might differ in terms of the ease of flap lifting. There is currently debate over the benefits of laser surface ablation versus LASIK surgery. Our goal as refractive surgeons is to provide patients with the best visual and refractive outcomes while minimizing the risks associated with each procedure. Being able to create predictably thin corneal flaps while eliminating unexpected thick flaps5,25 that increase the risk for ectasia26,27 is a major benefit to the LASIK procedure. It has been shown that mechanically created LASIK flaps biomechanically destabilize the cornea.28,29 Recent unpublished evidence suggests that if a LASIK flap could be created with a stromal thickness component of 30 to 60 mm, thereby creating a total corneal flap thickness of 80 to 100 mm, the biomechanics of the cornea would be virtually indistinguishable from those of a cornea after photorefractive keratectomy (PRK) (J. Marshall, FRCS, Wound Healing and Biomechanics of Corneal Flap Creation, presented at the XXIV Congress of the European Society of Cataract & Refractive Surgeons, London, England, September 2006). Creating such a thin corneal flap may increase the risk that the laser-created gas bubbles will break through the remaining anterior corneal stroma through Bowmans layer and the epithelium, inhibiting the incoming femtosecond laser beams and thus creating incomplete corneal flaps. By decreasing the

Figure 7. Transmission electron microscopy of the interface in specimen 2. The interface sides are smooth. A tissue bridge 10 mm high appears, like a stalagmite, in the middle of the figure. The raster energy was 0.7 mJ and the spot/line separation, of 9 mm 9 mm (bar Z 10 mm).

Figure 8. Transmission electron microscopy of the interface in specimen 3. Both sides of the interface are smooth. The nub of a tissue bridge of approximately 2 mm, simulating a stalactite, is seen. An intact keratocyte is seen adjacent to bottom of the interface. The raster energy was 0.4 mJ and the spot/line separation, 7 mm 7 mm (bar Z 2 mm).

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while matching the biomechanical advantages of surface laser ablation. Under these circumstances, the advantages of LASIK and PRK will blend into a single procedure that carries the benefits of both.

REFERENCES
1. Pallikaris IG, Papatzanaki ME, Siganos DS, Tsilimbaris MK. A corneal flap technique for laser in situ keratomileusis; human studies. Arch Ophthalmol 1991; 109:16991702 2. Solomon KD, Donnenfeld ED, Sandoval HP, et al. Flap-thickness accuracy: comparison of six microkeratome models; Flap-Thickness Study Group. J Cataract Refract Surg 2004; 30:964977 3. Duffey RJ. Thin flap laser in situ keratomileusis: flap dimensions with the Moria LSK-one manual microkeratome using the 100 mm head. J Cataract Refract Surg 2005; 31:11591162 4. Liu K-Y, Lam DSC. Direct measurement of microkeratome gap width by electron microscopy. J Cataract Refract Surg 2000; 27:924927 5. Flanagan GW, Binder PS. Precision of flap measurements for laser in situ keratomileusis in 4428 eyes. J Refract Surg 2003; 19:113123 JL. Confocal micros6. Javaloy J, Vidal MT, Ruiz-Moreno JM, Alio copy of disposable and nondisposable heads for the Moria M2 microkeratome. J Refract Surg 2006; 22:2833 7. Reinstein DZ, Sutton HFS, Srivannaboon S, et al. Evaluating microkeratome efficacy by 3D corneal lamellar flap thickness accuracy and reproducibility using Artemis VHF digital ultrasound arc-scanning. J Refract Surg 2006; 22:431440 8. Krueger RR, Quantock AJ, Juhasz T, et al. Ultrastructure of picosecond laser intrastromal photodisruption. J Refract Surg 1996; 12:607612 9. Nordan LT, Slade SG, Baker RN, et al. Femtosecond laser flap creation for laser in situ keratomileusis: six month follow-up of initial U.S. clinical series. J Refract Surg 2003; 19:814 10. Sarayba MA, Ignacio TS, Binder PS, Tran DB. Comparative study of stromal bed quality by using mechanical, IntraLase femtosecond 15- and 30-kHz microkeratomes. Cornea 2007; 26:446451 11. Sarayba MA, Ignacio TS, Tran DB, Binder PS. A 60 kHz IntraLase femtosecond laser creates a smoother LASIK stromal bed surface compared to a Zyoptix XP mechanical microkeratome in human donor eyes. J Refract Surg 2007; 23: 331337 12. Lim T, Yang S, Kim MJ, Tchah H. Comparison of the IntraLase femtosecond laser and mechanical microkeratome for laser in situ keratomileusis. Am J Ophthalmol 2006; 141:833839 13. Kezirian GM, Stonecipher KG. Comparison of the IntraLase femtosecond laser and mechanical keratomes for laser in situ keratomileusis. J Cataract Refract Surg 2004; 30:804811 14. Durrie DS, Kezirian GM. Femtosecond laser versus mechanical keratome flaps in wavefront-guided laser in situ keratomileusis; prospective contralateral eye study. J Cataract Refract Surg 2005; 31:120126 15. Tran DB, Sarayba MA, Bor T, et al. Randomized prospective clinical study comparing induced aberrations with IntraLase and Hansatome flap creation in fellow eyes; potential impact on wavefront-guided laser in situ keratomileusis. J Cataract Refract Surg 2005; 31:97105 16. Juhasz T, Kastis GA, Suarez C, et al. Time-resolved observations of shock waves and cavitation bubbles generated by femtosecond laser pulses in corneal tissue and water. Lasers Surg Med 1996; 19:2331

Figure 9. Transmission electron microscopy of the interface in specimen 4. Both sides of the interface are smooth. A nub of a tissue bridge of approximately 2 mm, simulating a stalactite, is seen on the posterior surface of the interface. Keratocytes that appear to be partially injured are seen on both sides of the interface. The raster energy was 0.4 mJ and the spot/line separation, 7 mm 7 mm (bar Z 10 mm).

spot/line separation of the laser ablation and the energies required for those spots, one can decrease the interface gas pressure and thereby decrease the risk for this so-called gas-breakthrough phenomenon. Current IntraLase software limits the surgeon from attempting to create a flap less than 90 mm flap thick. Because the standard deviation of IntraLase-created flaps using the current software version varies between 12 mm and 15 mm,22,23 2 standard deviations could place the interface at 60 mm; 3 standard deviations places the interface at 45 mm, which is within Bowmans layer. By reducing the spot/line separation of the femtosecond laser ablation and the associated energies, it may be possible to reduce the standard deviation of flap thickness, allowing the surgeon to safely create a thin corneal flap with excellent biomechanical stability, which is now termed sub-Bowmans keratomileusis (D.S. Durrie, et al., September 9, 2006, unpublished data). This study suggests that the benefit of increased speed permits decreased spot and line separations and decreased raster energy without significantly increasing procedure time. The potential for increased predictability of flap dimensions with lesser laser energies may provide surgeons with the proper tool to safely create a LASIK corneal flap that carries all the benefits over the bladed microkeratome-created flap

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nther T, Asiyo-Vogel M, Birngruber R. Factors deter17. Vogel A, Gu mining the refractive effects of intrastromal photorefractive keratectomy with the picosecond laser. J Cataract Refract Surg 1997; 23:13011310 18. Loesel FH, Niemz MH, Bille JF, Juhasz T. Laser-induced optical breakdown on hard and soft tissues and its dependence on the pulse duration: experiment and model. IEEE J Quant Electron 1996; 32:17171722 19. Kurtz RM, Elner V, Liu X, et al. Plasma-mediated ablation of biological tissue with picosecond and femtosecond laser pulses. In: Jacques SL, ed, Laser-Tissue Interaction VIII. Proceedings SPIE 2975. Bellington, WA, SPIE, 1997; 192200 20. Krueger RR, Juhasz T, Gualano A, Marchi V. The picosecond laser for nonmechanical laser in situ keratomileusis. J Refract Surg 1998; 14:467469 21. Binder PS. Flap dimensions created with the IntraLase FS laser. J Cataract Refract Surg 2004; 30:2632 22. Talamo JH, Meltzer J, Gardner J. Reproducibility of flap thickness with IntraLase FS and Moria LSK-1 and M2 microkeratomes. J Refract Surg 2006; 22:556561 23. Binder PS. One thousand consecutive IntraLase laser in situ keratomileusis flaps. J Cataract Refract Surg 2006; 32:962969 24. Stonecipher K, Dishler J, Ignacio TS, Binder PS. Transient light sensitivity after femtosecond laser flap creation: clinical findings and management. J Cataract Refract Surg 2006; 32:9194

25. Giledi O, Daya SM. Unexpected flap thickness in laser in situ keratomileusis. J Cataract Refract Surg 2003; 29:18251826 26. Flanagan G, Binder PS. Estimating residual stromal thickness before and after laser in situ keratomileusis. J Cataract Refract Surg 2003; 29:16741683 27. Binder PS, Lindstrom RL, Stulting RD. Keratoconus and corneal ectasia after LASIK [letter]. J Cataract Refract Surg 2005; 31:20352037; reply by E Donnenfeld, et al., 20372038 28. Qazi MA, Roberts CJ, Mahmoud AM, Pepose JS. Topographic and biomechanical differences between hyperopic and myopic laser in situ keratomileusis. J Cataract Refract Surg 2005; 31:4860 29. Twa MD, Roberts C, Mahmoud AM, Chang JS Jr. Response of the posterior corneal surface to laser in situ keratomileusis for myopia. J Cataract Refract Surg 2005; 31:6171

First author: Perry S. Binder, MS, MD Private practice, San Diego, and IntraLase, Inc., Irvine, California, USA

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