Light microscopy and scanning electron microscopy analysis of rigid curved interface femtosecond laser assisted and manual anterior capsulotomy

Transcription

1 LABORATORY SCIENCE Light microscopy and scanning electron microscopy analysis of rigid curved interface femtosecond laser assisted and manual anterior capsulotomy Marko Ostovic, MD, Oliver K. Klaproth, Dipl.-Ing. (FH), Fritz H. Hengerer, MD, PhD, Wolfgang J. Mayer, MD, FEBO, Thomas Kohnen, MD, PhD, FEBO PURPOSE: To study the microanatomic edge structures of anterior lens capsule specimens derived from manual and femtosecond laser assisted SETTING: Department of Ophthalmology, Goethe-University, Frankfurt, Germany. DESIGN: Experimental study. METHODS: Of 60 eyes with lens removal and intraocular lens implantation, 30 received a manual capsulotomy and 30 received a femtosecond laser assisted capsulotomy (Lensx, rigid curved interface, pulse energy 15 mj, spot separation 4 mm, layer separation 3 mm). After anterior capsule removal, tissues were immediately fixed in 4.5% formalin. Approximately 30 minutes after fixation, the tissues were removed from the fixation containers and air dried for at least 2 hours. Fifteen capsules in each group had further staining for light microscopy (LM). The surface of the capsulotomy edge was the primary focus of LM and scanning electron microscopy (SEM). Cell configuration, capsule shape, and abnormalities were evaluated. RESULTS: Subjective LM and SEM analysis showed smooth edges at all magnifications, no cell destruction, and cells up to the cutting edge in the manual capsulotomy group. Light microscopy demonstrated almost continuous anterior capsule incisions of the femtosecond laser assisted capsulotomy, a prominent demarcation line along the cutting edge, and several tags and bridges. Scanning electron microscopy showed microgrooves and valley- and mountain-like structures as signs of the photodisruption process. CONCLUSION: Compared with manual procedures, curved, rigid interface femtosecond laser assisted capsulotomy specimens using 15 mj pulse energy showed tags, bridges, rougher edges, and demarcation lines on the capsulotomy edges on SEM but subjectively estimated a more round shape on LM. Financial Disclosures: Mr. Klaproth received travel reimbursements and/or lecture fees from Alcon Laboratories, Inc., Rayner Intraocular Lenses Ltd., and Oculus GmbH. Dr. Kohnen received travel reimbursements, grant support, and/or lecture fees from Alcon Laboratories, Inc., Abbott Medical Optics, Inc., Bausch & Lomb, Carl Zeiss Meditec AG, Neoptics AG, Rayner Intraocular Lenses Ltd., and Schwind eye-tech-solutions GmbH and Co. KG; he is a consultant to Alcon Laboratories, Inc., Carl Zeiss Meditec AG, Rayner Intraocular Lenses Ltd., and Schwind eyetech-solutions GmbH and Co. KG. No other author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2013; 39: Q 2013 ASCRS and ESCRS For many years, manual continuous curvilinear capsulotomy (CCC) has been the method of choice for opening the anterior capsule in cataract surgery or refractive lens exchange. It is performed free hand with a capsule forceps or needles. 1 Although a highly skilled surgeon can manage a very precise and repeatable result, an optimum capsulotomy cannot be achieved in every patient. A precise and Q 2013 ASCRS and ESCRS Published by Elsevier Inc /$ - see front matter

2 1588 LABORATORY SCIENCE: LIGHT MICROSCOPY AND SEM OF FEMTOSECOND AND MANUAL CAPSULOTOMIES well-performed capsulotomy can improve the steps of cataract extraction, such as lens fragmentation, hydrodissection, and lens positioning, 2 4 and reduce complications, such as posterior capsule rupture. 5 Recently, femtosecond lasers became available for performing different steps of cataract surgery; that is, capsulotomy, lens fragmentation, and corneal incisions. 6 9 Studies 6,10 have shown that capsulotomies performed with a femtosecond laser have a repeatable, precise size and centration. Furthermore, they also show no tears. As a result, better intraocular lens (IOL) centration and less IOL tilt can be achieved. Symmetric capsulotomies with 360-degree overlap of the rim of the IOL optic can lead to better IOL centration and therefore better refractive results. 2,11 13 Appropriately sized and precise anterior capsulotomies maximize the performance, especially of premium IOLs, such as toric or multifocal models. 6,9 Sanders et al. 14 found that the smaller the capsulotomy, the greater the probability of a significantly reduced IOL shift due to capsule fibrosis. Increased optical quality and reduced internal aberrations were measured in eyes with femtosecond laser assisted 15 In a study by Trivedi et al., 16 scanning electron microscopy (SEM) showed evidence that smoother capsule edges led to stronger Friedman et al. 6 showed that decreasing laser pulse energy led to a decrease in the strength of the capsulotomy. Furthermore, SEM showed microgrooves on the capsule edges produced by the laser. Therefore, the purpose of our study was to assess the microanatomic structures of anterior human lens capsule specimens after manual and femtosecond laser assisted capsulotomy. MATERIALS AND METHODS Sixty capsulotomy specimens divided into 2 groups (30 in manual CCC group; 30 in femtosecond laser assisted group) were collected during lens removal surgery and immediately preserved in 4.5% formalin solution for further analysis. Only completely extracted and mounted specimens were used. Submitted: June 8, Final revision submitted: June 24, Accepted: July 11, From the Departments of Ophthalmology, Goethe-University (Ostovic, Klaproth, Hengerer, Kohnen), Frankfurt am Main, and Ludwig-Maximilians-University (Mayer), Munich, Germany. Corresponding author: Thomas Kohnen, MD, PhD, FEBO, Department of Ophthalmology, Goethe-University, Theodor-Stern-Kai 7, Frankfurt am Main, Germany. kohnen@em.unifrankfurt.de. Surgical Technique The standard phacoemulsification, manual capsulotomy, and femtosecond laser assisted capsulotomy procedures were performed under topical anesthesia of oxybuprocaine hydrochloride (Conjucain EDO) eyedrops by the same surgeon (T.K.). All surgeries were uneventful. Manual capsulotomies were created using an Utrata forceps (Geuder, Heidelberg, Germany) and centripetal forces with regrasping maneuvers. The femtosecond laser assisted capsulotomies were performed using the LenSx platform (Alcon Laboratories, Inc.). The infrared femtosecond laser has a repetition rate of 33 khz, pulse width of 600 to 800 fs, and central laser wavelength of 1030 nm. In all specimens, the laser energy setting was 15 mj with a spot separation of 6 mm and a layer separation of 3 mm. The rigid interface surface had a diameter of 10.8 mm and a curvature of 8.3 mm. The depth and coordinates of the femtosecond laser assisted capsulotomies were determined with the live optical coherence tomography integrated into the laser system. The patient interface consists of an applanation lens, suction ring, and tubing. After docking was performed, the cornea was applanated and suction activated. Light Microscopy Images were captured and analyzed with a Leica/Reichert Univar microscope with Leica DFC 280 camera system (Leica Microsystems Imaging Solutions Ltd.) using different magnifications (2 to20). Staining For Masson-Goldner trichrome staining, the anterior capsules were deparaffinized, rehydrated through different concentrations of alcohol, differentiated in phosphomolybdic phosphotungstic and acetic acid, washed in distilled water, and finally embedded in xylole. Scanning Electron Microscopy Coating the specimens for SEM was performed with 25 nm of gold and examined with SEM at 30 KV. The SEM images of the complete capsulotomy were captured with a Hitachi S-500 scanning electron microscope (Hitachi High- Technologies Corp.), with further focus on 3 random areas for all specimens. Primary and secondary endpoints of the study were cell structure, tears in the capsule edge, configuration of the nuclei and cell structure, and abnormalities of the RESULTS Manually Performed Capsulotomies Lightmicroscopy(LM)showedintactcellnuclei and plasma; cells had intact cellular borders and were arranged directly at the edge of the cut (Figure 1). Of note, there was no demarcation line when the manual capsulotomies were analyzed. The only tears found at the edge were those acquired using the CCC method.

3 LABORATORY SCIENCE: LIGHT MICROSCOPY AND SEM OF FEMTOSECOND AND MANUAL CAPSULOTOMIES 1589 Figure 1. Constant configuration with intact cell structure of the manual capsulorhexis edge (CCC; LM 10 magnification; MassonGoldner trichrome staining). Figure 2. Visible anterior capsule fibers (CCC; SEM magnification). Scanning electron microscopy analysis showed constant configuration of the manual capsulotomy edge at all magnifications (from 30 to ). With magnifications of 3000 and higher, the individual fibers of the capsule could be identified (Figure 2). No tags, bridges, or tears were seen in manual The mean time for creating capsulotomies was 1.9 G 0.6 (SD) seconds. At magnifications higher than 20, all 30 capsules showed a demarcation line along the cutting edge. This area had a width of approximately 60 mm. Along the inner part of the line, in the direction of the capsule center, the cell configuration showed irregularities (Figure 3). The cell nuclei and the cell plasma were partly destroyed and spread along the demarcation line. The femtosecond laser assisted capsulotomies also showed multiple tags, bridges, and stray impressions, even at some distance from the cutting edge (Figure 4). The tags had variable distances, with the longest one being more than 1.0 mm (Figure 5). At 100 magnification in SEM, individual laser spots and grooves could be identified (Figure 6). At magnification, frayed tissue became visible. Here, valley- and mountain-like structures could be seen. With increasing magnification, the edges of the femtosecond laser - assisted capsulotomies showed a Figure 3. Cellular irregularities and visible demarcation line along the cutting edge (femtosecond laser assisted capsulotomy; LM 20 magnification; Masson-Goldner trichrome staining). Figure 4. Tags along the cutting edge (femtosecond laser assisted capsulotomy; LM 4 magnification; Masson-Goldner trichrome staining). Femtosecond Laser Assisted Capsulotomies

4 1590 LABORATORY SCIENCE: LIGHT MICROSCOPY AND SEM OF FEMTOSECOND AND MANUAL CAPSULOTOMIES Figure 5. Longest tag of 1.7 mm (femtosecond laser assisted capsulotomy; LM 4 magnification; Masson-Goldner trichrome staining). Figure 6. Visible laser spots (femtosecond laser assisted capsulotomy; SEM 100 magnification). Until now, analysis of femtosecond laser assisted lens surgery mainly focused on the macroscopic shape and centration of capsulotomies, the capsular bag's postoperative tensile strength, or clinical implications, such as postoperative refractive outcomes.6 9,11 15 This study evaluated the fine microscopic structure of capsulotomy edges using LM and SEM. Tags and bridges could not be related to the laser beam steering because we made the assumption that the laser itself cuts perfectly regular patterns. Therefore, we believe that the tags and bridges are caused by minimal torsional movements of the eye, which we observed during surgery, even with the eye fixated by limbal conjunctival suction of the patient interface. This effect may be reduced when a liquid immersion interface is used, as described by Talamo et al.,17 who found that the eye drifts were lower using a liquid optical immersion interface, and by reducing pulse energies, as shown by Friedman et al.6 and Palanker et al.13 The most likely reason for the straying is the design of the patient interface.17,18 All femtosecond laser assisted lens removal procedures were performed with a rigid, curved patient interface, which applanates the cornea. Pressing the cornea against a rigid surface with a curvature different from the cornea's natural shape causes deformations of the cornea, Figure 7. Sawtooth pattern along the demarcation line (femtosecond-laser-assisted capsulotomy, SEM, 3000 magnification). Figure 8. Tag (femtosecond laser assisted capsulotomy; SEM 1000 magnification). sawtooth pattern with individual grooves on the cut edge (Figure 7). Tags were still clearly visible (Figure 8). DISCUSSION

5 LABORATORY SCIENCE: LIGHT MICROSCOPY AND SEM OF FEMTOSECOND AND MANUAL CAPSULOTOMIES 1591 resulting in unwanted folds in the posterior surface. Such folds may not affect the laser focusing inside the stroma; if this is the case, the effect is not a significant issue in the corneal flap cutting during refractive corneal surgery. However, for the beam focused several millimeters posterior to the cornea, the effect of the corneal folds might be critically important. Possible results of this are damage of near structures in the patient's eye, such as iris micro injuries in small pupils. Talamo et al. 17 recently described changes between 2 interfaces and stated that curved contact interfaces can lead to incomplete capsulotomies during laser-assisted cataract surgery. A major finding in this study was the clear demarcation line in the femtosecond laser assisted capsulotomy edges, which confirms an effect of the laser on the surrounding tissue. Previous studies 19,20 showed that photodisruption was not responsible for significant damage to surrounding structures. It is known that in general, femtosecond lasers cut tissue by vaporizing it, creating plasma and, finally, a cavitation bubble, which expands and collapses, thus separating tissue. The connection of these bubbles then creates the cut surface. 21 This demarcation line could be the result of this expanding cavitation bubble in combination with the chosen relatively high pulse energy of 15 mj, which might lead to displacement of cells and destruction of cell nuclei close to the cutting edge. A study by Trivedi et al. 16 found that the manual technique produced the most extensible capsulotomy; however, the study was performed using a porcine model, as were the studies of Friedman et al. 6 and Auffarth et al. 22 We are aware that we used only 1 energy pulse level (15 mj) for morphologic analyses. Our focus was not to study capsulotomy strength but rather microanatomic changes. Although the femtosecond laser seems to be a promising and safe technology in cataract and refractive lens surgery, side effects from the laser beams on the tissue have to be considered. Our fine structural analysis showed a certain amount of straying of the laser spots as well as tags and bridges at the capsulotomy edges and a demarcation line of approximately 60 mm along the cut. We presume that by lowering the pulse energy and/or using a liquid immersion/soft interface, as described by Talamo et al., 17 the demarcation line, and therefore the impact on surrounding tissue, could be reduced without considering the effect on capsulotomy strength. Further studies of the relationship between pulse energy, patient interface design, and capsule burst strength will help elucidate the optimum parameters for laser capsulotomy creation. WHAT WAS KNOWN Femtosecond lasers produce repeatable and precise The design of the patient interface is important to the outcome of the procedure. WHAT THIS PAPER ADDS Photodisruption of the anterior lens capsule with a central laser wavelength of 1030 nm and maximum pulse energy of 15 mj had an effect on the surrounding tissue. Rigid, curved interface 15 mj pulse energy femtosecond laser acquired capsulotomies showed tags, bridges, and demarcation lines along the cut and rough edges with valley- and mountain-like structures on SEM. REFERENCES 1. Gimbel HV, Neuhann T. Development, advantages, and methods of the continuous circular capsulorhexis technique. J Cataract Refract Surg 1990; 16: Szigeti A, Kranitz K, Takacs AI, Mihaltz K, Knorz MC, Nagy ZZ. Comparison of long-term visual outcome and IOL position with a single-optic accommodating IOL after 5.5- or 6.0-mm femtosecond laser capsulotomy. J Refract Surg 2012; 28: Martin KR, Burton RL. The phacoemulsification learning curve: per-operative complications in the first 3000 cases of an experienced surgeon. Eye 2000; 14: Available at: pdf. Accessed June 28, Olali CA, Ahmed S, Gupta M. Surgical outcome following breach rhexis. Eur J Ophthalmol 2007; 17: Kohnen T. Kapsel- und Zonularupturen als Komplikation der Kataraktoperation mit Phakoemulsifikation [Inaugural- Dissertation]. Bonn, Germany, Fachbereiches Humanmedizin der Friedrich-Wilhelms Universit at Bonn, Friedman NJ, Palanker DV, Schuele G, Andersen D, Marcellino G, Seibel BS, Batlle J, Feliz R, Talamo JH, Blumenkranz MS, Culbertson WW. Femtosecond laser capsulotomy. J Cataract Refract Surg 2011; 37: Bali SJ, Hodge C, Lawless M, Roberts TV, Sutton G. Early experience with the femtosecond laser for cataract surgery. Ophthalmology 2012; 119: Roberts TV, Lawless M, Chan CC, Jacobs M, Ng D, Bali SJ, Hodge C, Sutton G. Femtosecond laser cataract surgery: technology and clinical practice. Clin Exp Ophthalmol 2012; 41: He L, Sheehy K, Culbertson W. Femtosecond laser-assisted cataract surgery. Curr Opin Ophthalmol 2011; 22: Baumeister M, Neidardt B, Strobel J, Kohnen T. Tilt and centration of three-piece foldable high-refractive silicone and hydrophobic acrylic intraocular lenses with 6-mm optics in an intraindividual comparison. Am J Ophthalmol 2005; 140: KranitzK, Mihaltz K, Sandor GL, Takacs A, Knorz MC, Nagy ZZ. Intraocular lens tilt and decentration measured by Scheimpflug camera following manual or femtosecond laser-created continuous circular capsulotomy. J Refract Surg 2012; 28:

6 1592 LABORATORY SCIENCE: LIGHT MICROSCOPY AND SEM OF FEMTOSECOND AND MANUAL CAPSULOTOMIES 12. Nagy Z, Takacs A, Filkorn T, Sarayba M. Initial clinical evaluation of an intraocular femtosecond laser in cataract surgery. J Refract Surg 2009; 25: Palanker DV, Blumenkranz MS, Andersen D, Wiltberger M, Marcellino G, Gooding P, Angeley D, Schuele G, Woodley B, Simoneau M, Friedman NJ, Seibel B, Batlle J, Feliz R, Talamo J, Culbertson W. Femtosecond laser-assisted cataract surgery with integrated optical coherence tomography. Sci Transl Med 2010; 2:58ra85. Available at: edu/wpalanker/publications/fs_laser_cataract.pdf. Accessed June 28, Sanders DR, Higginbotham RW, Opatowsky IE, Confino J. Hyperopic shift in refraction associated with implantation of the single-piece Collamer intraocular lens. J Cataract Refract Surg 2006; 32: Mihaltz K, Knorz MC, Alio JL, Takacs AI, Kranitz K, Kovacs I, Nagy ZZ. Internal aberrations and optical quality after femtosecond laser anterior capsulotomy in cataract surgery. J Refract Surg 2011; 27: Trivedi RH, Wilson ME Jr, Bartholomew LR. Extensibility and scanning electron microscopy evaluation of 5 pediatric anterior capsulotomy techniques in a porcine model. J Cataract Refract Surg 2006; 32: Talamo JH, Gooding P, Angeley D, Culbertson WW, Schuele G, Andersen D, Marcellino G, Essock-Burns E, Batlle J, Feliz R, Friedman NJ, Palanker D. Optical patient interface in femtosecond laser-assisted cataract surgery: contact corneal applanation versus liquid immersion. J Cataract Refract Surg 2013; 39: Kohnen T. Interface for femtosecond laser-assisted lens surgery [editorial]. J Cataract Refract Surg 2013; 39: Vogel A, Noack J, Nahen K, Theisen1 D, Busch1 S, Parlitz U, Hammer DX, Noojin GD, Rockwell BA, Birngruber R. Energy balance of optical breakdown in water at nanosecond to femtosecond time scales. Appl Phys B Lasers Opt 1999; 68: Available at: uploads/tx_wapublications/vogel 1999_Appl._Phys._Energy_ balance_of_optical_breakdown_in_water_at_nanosecond_to_ femtosecond_time_scales.pdf. Accessed June 28, Vogel A, Noack J, H uttman G, Paltauf G. Mechanisms of femtosecond laser nanosurgery of cells and tissues. Appl Phys B Lasers Opt 2005; 81: Available at: Documento1.pdf. Accessed June 28, Kurtz RM, Liu X, Elner VM, Squier JA, Du D, Mourou GA. Photodisruption in the human cornea as a function of laser pulse width. J Refract Surg 1997; 13: Auffarth GU, Reddy KP, Ritter R, Holzer MP, Rabsilber TM. Comparison of the maximum applicable stretch force after femtosecond laser-assisted and manual anterior capsulotomy. J Cataract Refract Surg 2013; 39: First author: Marko Ostovic, MD Department of Ophthalmology, Goethe-University, Frankfurt am Main, Germany