Evaluating the effect of pulse energy on femtosecond laser trabeculotomy (FLT) outflow channels for glaucoma treatment in human cadaver eyes.

BACKGROUND AND OBJECTIVES: Femtosecond laser trabeculotomy (FLT) creates aqueous humor outflow channels through the trabecular meshwork (TM) and is an emerging noninvasive treatment for open-angle glaucoma. The purpose of this study is to investigate the effect of pulse energy on outflow channel creation during FLT. MATERIALS AND METHODS: An FLT laser (ViaLase Inc.) was used to create outflow channels through the TM (500 µm wide by 200 µm high) in human cadaver eyes using pulse energies of 10, 15, and 20 µJ. Following treatment, tissues were fixed in 4% paraformaldehyde. The channels were imaged using optical coherence tomography (OCT) and assessed as full thickness, partial thickness, or not observable. RESULTS: Pulse energies of 15 and 20 µJ had a 100% success rate in creating full-thickness FLT channels as imaged by OCT. A pulse energy of 10 µJ resulted in no channels (n = 6), a partial-thickness channel (n = 2), and a full-thickness FLT channel (n = 2). There was a statistically significant difference in cutting widths between the 10 and 15 µJ groups (p < 0.0001), as well as between the 10 and 20 µJ groups (p < 0.0001). However, there was no statistically significant difference between the 15 and 20 µJ groups (p = 0.416). CONCLUSIONS: Fifteen microjoules is an adequate pulse energy to reliably create aqueous humor outflow channels during FLT in human cadaver eyes. OCT is a valuable tool when evaluating FLT.

Nonlinear optical crosslinking (NLO CXL) for correcting refractive errors.

Ultraviolet A (UVA) light-based photoactivation of riboflavin (Rf) to induce corneal crosslinking (CXL) and mechanical stiffening is now a well-known treatment for corneal ectasia and Keratoconus that is being used in a topographically guided photorefractive intrastromal CXL (PiXL) procedure to treat low degrees of refractive errors. Alternative approaches for non-invasive treatment of refractive errors have also been proposed that use femtosecond lasers (FS) that provide much faster, more precise, and safer results than UVA CXL. One such treatment, nonlinear optical crosslinking (NLO CXL), has been able to replicate the effects of UVA CXL, while producing a smaller area of cellular damage and requiring a shorter procedure time. Unlike UVA CXL, the treatment volume of NLO CXL only occurs within the focal volume of the laser, which can be placed at any depth and scanned into any pattern for true topographically guided refractive correction. This review presents our experience with using FS lasers to photoactivate Rf and perform highly controlled corneal CXL that leads to mechanical stiffening and changes in corneal shape.

Collagen fiber crimping following in vivo UVA-induced corneal crosslinking.

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 µm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ±â€¯3.4% and 47.7 ±â€¯7.6% of the corneal thickness at one and three months. There was a significant (P < 0.05) reduction in CFC in the CAF region in all eyes averaging 1.007 ±â€¯0.006 and 1.009 ±â€¯0.005 in one and three month samples compared to 1.017 ±â€¯0.04 and 1.016 ±â€¯0.06 for controls. These results indicate that there is a significant reduction in collagen crimping following UVA CXL of approximately 1%. One possible explanation for this loss of crimping could be shortening of the collagen fibers over the CXL region.

Axial mechanical and structural characterization of keratoconus corneas.

PURPOSE: Previous studies indicate that there is an axial gradient of collagen lamellar branching and anastomosing leading to regional differences in corneal tissue stiffness that may control corneal shape. To further test this hypothesis we have measured the axial material stiffness and quantified the collagen lamellar complexity in ectatic and mechanically weakened keratoconus corneas (KC). METHODS: Acoustic radiation force elastic microscopy (ARFEM) was used to probe the axial mechanical properties of the cone region of three donor KC buttons. 3 Dimensional second harmonic generation microscopy (3D-SHG) was used to qualitatively evaluate lamellar organization in 3 kC buttons and quantitatively measure lamellar branching point density (BPD) in a separate KC button that had been treated with epikeratophakia (Epi-KP). RESULTS: The mean elastic modulus for the KC corneas was 1.67 ±â€¯0.44 kPa anteriorly and 0.970 ±â€¯0.30 kPa posteriorly, substantially below that previously measured for normal human cornea. 3D-SHG of KC buttons showed a simplified collagen lamellar structure lacking noticeable angled lamellae in the region of the cone. BPD in the anterior, posterior, central and paracentral regions of the KC cornea were significantly lower than in the overlying Epi-KP lenticule. Additionally, BPD in the cone region was significantly lower than the adjacent paracentral region in the KC button. CONCLUSIONS: The KC cornea exhibits an axial gradient of mechanical stiffness and a BPD that appears substantially lower in the cone region compared to normal cornea. The findings reinforce the hypothesis that collagen architecture may control corneal mechanical stiffness and hence corneal shape.

Evaluation of the mechanical properties of the anterior lens capsule following femtosecond laser capsulotomy at different pulse energy settings.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule opening performed with femtosecond laser capsulotomy at different energy settings in ex vivo porcine anterior lens capsule specimens. METHODS: Twenty-five fresh porcine eyes per group were included in the study. Femtosecond laser capsulotomy was performed with three different pulse energy levels: 2 µJ (low energy group), 5 µJ (intermediate energy group), and 10 µJ (high energy group). The capsule openings were stretched with universal testing equipment until they ruptured. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy. RESULTS: The high energy group had significantly lower rupture force (108 ± 14 mN) compared to the intermediate energy group (118 ± 10 mN) (P < .05) and low energy group (119 ± 11 mN) (P < .05), but the difference between the intermediate energy and low energy groups was not significant (P = .9479). The high energy group had significantly lower circumference stretching ratio (144% ± 3%) compared to the intermediate energy group (148% ± 3%) (P < .05) and low energy group (148% ± 3%) (P < .05), but the difference between the intermediate energy group and low energy group was not significant (P = .9985). Scanning electron microscopy images showed that the edge was only serrated with low and intermediate energy, but additional signs of collagen melting and denaturation were observed at high energy. CONCLUSIONS: Anterior capsule openings created at a high energy level were slightly weaker and less extensible than those created at low or intermediate levels, possibly due to the increased thermal effect of photo-disruption.

Comparison of the mechanical properties of the anterior lens capsule following manual capsulorhexis and femtosecond laser capsulotomy.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule openings performed with the continuous curvilinear capsulorhexis (CCC) technique and femtosecond laser capsulotomy (FLC) in ex vivo porcine lens capsule specimens. METHODS: Fresh porcine eyes were included in the study (CCC group, n = 50; FLC group, n = 30). The capsule openings were stretched with universal testing equipment until they ruptured. The rupture force and circumference stretching ratio were evaluated. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy (SEM). RESULTS: The average rupture force was higher in the CCC group (median: 155 mN; interquartile range [IQR]: 129 to 201 mN; range: 71 to 294 mN) than in the FLC group (median: 119 mN; IQR: 108 to 128 mN; range: 91 to 142 mN) (P < .01, Mann-Whitney U test). The average circumference stretching ratio in the CCC group was greater (median: 150%; IQR: 146% to 156%; range: 136% to 161%) than in the FLC group (median: 148%; IQR: 145% to 150%; range: 141% to 154%) (P = .0468, Mann-Whitney U test). When less than 71 mN, no capsular tear occurred in either group. When less than 91 mN, no capsular tear occurred in the FLC group, whereas at 91 mN, the probability of capsular tears was 9% for the CCC group. SEM examination found that the CCC group had smooth edges, whereas those of the FLC group were gently serrated. CONCLUSIONS: According to the current results in a porcine eye model, FLC had less average resistance to capsule tear than CCC, but the weakest openings were seen in the CCC group.

Modeling meibum secretion: Alternatives for obstructive Meibomian Gland Dysfunction (MGD).

PURPOSE: While changes in meibum quality are correlated with severity of meibomian gland dysfunction (MGD) and dry eye disease, little is known regarding the mechanics of meibum secretion. The purpose of this study was to develop a finite element model of meibum secretion and evaluate the effect of various factors that might impact meibum delivery to the ocular surface. METHODS: A finite element analysis in COMSOL 6.0 was used to simulate the flow of meibum within the gland's terminal excretory duct. Historical normal human meibum rheology data taken over the meibum melting range from fluid (35-40 °C) to solid (25-30 °C) were then used to calculate the minimum yield stress and plastic viscosity of meibum. The effects of meibum melting state, eyelid pressure and terminal duct diameter on meibum flow rates were then systematically investigated. RESULTS: The melting state of meibum from liquid to solid was associated with an increase in the minimum yield stress and plastic viscosity that caused an exponential decrease in meibum flow. Modeling also established that there was a linear correlation between meibum flow rate and eyelid pressure needed to express meibum and the 4th power of the terminal duct radius. CONCLUSIONS: Our results suggest that changes in the melting state of meibum from fluid to solid, as well as changes in the radius of the terminal excretory duct and the force exerted by the eyelid can lead to dramatic decreases in the flow of meibum. Together these findings suggest alternative mechanisms for meibomian gland obstruction.

Anterior segment OCT imaging after femtosecond laser cataract surgery.

PURPOSE: To report the anterior segment imaging characteristics after femtosecond laser assisted cataract surgery. METHODS: Cataract surgery was performed with the LenSx femtosecond laser (Alcon-LenSx Inc., Aliso Viejo, CA) in 40 eyes of 40 patients. The laser was programmed to perform a 4.5-mm capsulorhexis, a cross-pattern fragmentation of the nucleus, a 2.8-mm main incision, and a 1.0-mm side-port incision. The anterior segment was then analyzed using the Visante OCT anterior segment program (Zeiss-Meditec AG, Jena, Germany). RESULTS: The preoperatively set treatment parameters correlated well with the achieved results. For the capsulorhexis, the femtosecond laser cut was programmed to start 350 µm behind the anterior lens capsule and OCT measured 377 ± 55.3 µm. Nucleus fragmentation was programmed to start 750 µm in front of the posterior capsule and end 550 µm behind the anterior capsule, and OCT measured 794 ± 111 and 568 ± 147 µm, respectively. The diameter of the capsulorhexis measured by OCT was 4.54 ± 0.2 mm, compared to the 4.5 mm programmed. CONCLUSIONS: Anterior segment OCT imaging was able to detect the tissue changes within the lens after femtosecond laser capsulorhexis and nucleus fragmentation. The measured values correlated well with the planned treatment parameters.

A review of the epithelial and stromal effects of corneal collagen crosslinking.

Induced corneal collagen crosslinking and mechanical stiffening via ultraviolet-A photoactivation of riboflavin (UVA CXL) is now a common treatment for corneal ectasia and Keratoconus. Some effects of the procedure such as induced mechanical stiffening, corneal flattening, and cellular toxicity are well-known, but others remain more controversial. Authors report a variety of contradictory effects, and provide evidence based on individual results and observations. A full understanding of the effects of and mechanisms behind this procedure are essential to predicting its outcome. A growing interest in modifications to the standard UVA CXL protocol, such as transepithelial or accelerated UVA CXL, makes analyzing the literature as a whole more urgent. This review presents an analysis of both the agreed-upon and contradictory results reported and the various methods used to obtain them.

Iridocorneal angle imaging of a human donor eye by spectral-domain optical coherence tomography.

Iridocorneal angle (ICA) details particularly the trabecular meshwork (TM), Schlemm's canal (SC), and collector channels (CCs) play crucial roles in the regulation of the aqueous outflow in the eyes and are closely associated with glaucoma. Current clinical gonioscopy imaging provides no depth information, and studies of 3D high-resolution optical coherence tomography (OCT) imaging of these structures are limited. We developed a custom-built spectral-domain (SD-) OCT imaging system to fully characterize the angle details. Imaging of a human cadaver eye reveals the visibility of details in the TM/SC/CC region via a 'crossline' scanning and a series of image processing. This shows that ICA imaging can be used for preoperative glaucoma inspections in the clinical setting with the proposed prototype.

First-in-Human Safety Study of Femtosecond Laser Image-Guided Trabeculotomy for Glaucoma Treatment: 24-month Outcomes.

Purpose: Pilot study to evaluate adverse events and intraocular pressure (IOP)-lowering of a novel, noninvasive glaucoma procedure, femtosecond laser, image-guided, high-precision trabeculotomy (FLIGHT). Design: Prospective, nonrandomized, single-center, interventional, single-arm clinical trial. Participants: Eighteen eyes from 12 patients with open-angle glaucoma. Methods: Eighteen eyes from 12 patients underwent FLIGHT, creating a single channel measuring 500-µm wide by 200-µm high through the trabecular meshwork and into Schlemm's canal. Adverse events, IOP, and other parameters were evaluated out to 24 months. Main Outcome Measures: Outcomes were the rates and types of adverse events and the rate of postprocedure best-corrected visual acuity loss (≥ 2 lines) compared with baseline. Efficacy outcomes were reduction in mean intraocular pressure (IOP) with respect to baseline and the percentage of eyes with a ≥ 20% reduction in IOP. Results: Eighteen eyes from 12 patients were enrolled in the study; 11 patients (17 eyes) returned at 24 months. There were no serious adverse events related to the laser treatment. Well-defined channels were clearly visible at 24 months by gonioscopy and anterior segment OCT, with no evidence of closure. At 24 months, the mean IOP was reduced by 34.6% from 22.3 ± 5.5 to 14.5 ± 2.6 mmHg (P < 5e-5), with an average of 2.0 ± 1.2 hypotensive medications compared with 2.2 ± 1.1 at baseline (P = 0.22). Fourteen out of the 17 study eyes (82.3%) achieved a ≥ 20% reduction in IOP at 24 months when compared with baseline. Conclusion: The FLIGHT system demonstrated a favorable safety profile in this initial pilot study, with no device-related serious adverse events. The channels appeared patent at 24 months, indicating medium-term durability. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Femtosecond Laser Trabeculotomy in Perfused Human Cadaver Anterior Segments: A Novel, Noninvasive Approach to Glaucoma Treatment.

Purpose: The purpose of this study was to investigate femtosecond laser trabeculotomy (FLT) in a clinically relevant manner (i.e., delivering the surgical laser beam through the cornea of the intact, human anterior segment to create channels from the anterior chamber into the Schlemm's canal) and to investigate the effect of this treatment on intraocular pressure in perfused human anterior segments. Methods: Perfused human anterior segments (15 eyes) received either FLT treatment (n = 8) or a sham-treatment (n = 7). Intraocular pressure (IOP) in the perfused samples was recorded before and after treatment. Spectral domain optical coherence tomography, second harmonic generation imaging, and transmission electron microscopy were used to investigate the FLT channels. Results: The FLT group (n = 7, 1 eye excluded) had a statistically significant reduction in mean IOP of 20.2% from baseline after treatment (5.06 ± 1.46 mm Hg to 4.04 ± 1.63 mm Hg; P < 0.0005), whereas the control group (n = 7) remained statistically unchanged (7.72 ± 3.45 mm Hg to 7.78 ± 3.51 mm Hg; P < 0.71). Imaging confirmed that the channels traversed the entire trabecular meshwork into the Schlemm's canal. Conclusions: This study has provided the first direct evidence supporting the feasibility of clinically applicable, noninvasive femtosecond laser trabeculotomy for the treatment of glaucoma. Various imaging modalities revealed minimal collateral damage to adjacent issues. Translational Relevance: This work demonstrates noninvasive femtosecond laser trabeculotomy in a laboratory setting that is clinically relevant.

Intraocular Pressure Reduction by Femtosecond Laser Created Trabecular Channels in Perfused Human Anterior Segments.

Purpose: This study investigated the initial feasibility of using femtosecond laser trabeculotomy (FLT) to create open channels through the trabecular meshwork into Schlemm's canal to lower intraocular pressure (IOP) in a perfused anterior segment model. Methods: Human anterior segments (12 eyes) were assigned to either treatment (n = 6) or sham treatment (n = 6) groups. Both groups were perfused until a baseline IOP was recorded upon which a direct FLT treatment or a sham treatment was administered. IOP was recorded before and after the treatment. Spectral domain optical coherence tomography and second harmonic generation imaging we used to investigate the FLT channels. Results: In the FLT group, there was a significant mean decrease in the IOP of 22% compared with the pre-FLT IOP (7.13 ± 2.95 mm Hg to 5.34 ± 1.62 mm Hg; P < 0.05). In the control group, the post-sham IOP remained relatively unchanged compared with the pre-sham IOP (6.39 ± 3.69 mm Hg to 6.67 ± 4.12 mm Hg). Conclusions: The results of this study indicate that FLT treatment can significantly decrease the IOP in a perfusion model and may provide a potential noninvasive treatment option for primary open angle glaucoma. Translational Relevance: Investigating the use of femtosecond lasers for photodisrupting the trabecular meshwork can lead to a clinically relevant alternative to current glaucoma procedures.

Temperature increase in human cadaver retina during direct illumination by femtosecond laser pulses.

PURPOSE: Femtosecond lasers have been approved by the US Food and Drug Administration for ophthalmic surgery, including use in creating corneal flaps in LASIK surgery. During normal operation, approximately 50% to 60% of laser energy may pass beyond the cornea, with potential effects on the retina. As a model for retinal laser exposure during femtosecond corneal surgery, we measured the temperature rise in human cadaver retinas during direct illumination by the laser. METHODS: The temperature increase induced by a 150-kHz iFS Advanced Femtosecond Laser (Abbott Medical Optics) in human cadaver retinas was measured in situ using an infrared thermal imaging camera. To model the geometry of the eye during the surgery, an approximate 11x11-mm excised section of human cadaver retina was placed 17 mm behind the focus of the laser beam. The temperature field was observed in 10 cadaver retina samples at energy levels ranging from 0.4 to 1.6 microJ (corresponding approximately to surgical energies of 0.8 to 3.2 microJ per pulse). RESULTS: Maximal temperature increases up to 1.15 degrees C (corresponding to 3.2 microJ and 52-second illumination) were observed in the cadaver retina sections with little variation in temperature profiles between specimens for the same laser energy illumination. CONCLUSIONS: The commercial iFS Advanced Femtosecond Laser operating with pulse energies at approximately the lower limit of the range evaluated in this study would be expected to result in a 0.2 degrees C temperature increase and do not therefore present a safety hazard to the retina.

In vivo femtosecond laser subsurface scleral treatment in rabbit eyes.

BACKGROUND AND OBJECTIVES: The progression of glaucoma can be reduced or delayed by reducing intraocular pressure (IOP). The properties of femtosecond laser surgery, such as markedly reduced collateral tissue damage, coupled with the ability to achieve isolated subsurface surgical effects in the sclera, make this technology a promising candidate in glaucoma management. In this pilot study we demonstrate the in vivo creation of partial thickness subsurface drainage channels with the femtosecond laser in the sclera of rabbit eyes in order to increase aqueous humor (AH) outflow. STUDY DESIGN/MATERIALS AND METHODS: A femtosecond laser beam tuned to a 1.7 microm wavelength was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of one eye of each of the four rabbits included in this pilot study. IOP was measured before and 20 minutes after the laser treatment to evaluate the acute effect of the procedure. RESULTS: OCT images verified the creation of the partial thickness subsurface scleral channels in the eyes of the in vivo rabbits. Comparison of pre- and postoperative IOP measurements in treated and control eyes revealed a reduction in the intraocular pressure due to the increased rate of AH outflow resulted in by the presence of the partial thickness scleral channels. CONCLUSIONS: The creation of partial thickness subsurface drainage channels was demonstrated in the sclera of in vivo rabbit eyes with a 1.7 microm wavelength femtosecond laser. Reduction in IOP achieved by the partial thickness channels suggests potential utility in the treatment of elevated IOP.

Enhanced Transepithelial Riboflavin Delivery Using Femtosecond Laser-Machined Epithelial Microchannels.

Purpose: This study describes a femtosecond laser (FS) approach to machine corneal epithelial microchannels for enhancing riboflavin (Rf) penetration into the cornea prior to corneal crosslinking (CXL). Methods: Using a 1030-nm FS laser with 5- to 10-µJ pulse energy, the corneal epithelium of slaughterhouse rabbit eyes was machined to create 2-µm-diameter by 25-µm-long microchannels at a density of 100 or 400 channels/mm2. Rf penetration through the microchannels was then determined by applying 1% Rf in phosphate-buffered saline for 30 minutes followed by removal of the cornea and extraction from the central stromal button. Stromal Rf concentrations were then compared to those obtained using standard epithelial debridement or 0.01% benzalkonium chloride (BAK) to disrupt the epithelial barrier. Results: Microchannels formed using a 5-µJ/pulse at a density of 400 channels/mm2 achieved a stromal Rf concentration that was 50% of that achieved by removal of the corneal epithelium and imbibing with 1% Rf. Stromal Rf levels were also equal to that of debrided corneas soaked with 0.5% Rf, threefold higher than those soaked with 0.1% Rf, and twofold higher than corneas soaked in BAK without epithelial debridement. Organ culture of treated corneas showed a normal corneal epithelium following FS machining while BAK-treated corneas showed extensive epithelial and stromal damage at 24 hours posttreatment. Conclusions: FS corneal epithelial machining can be used to enhance penetration of Rf into the stroma for corneal CXL. Translational Relevance: The creation of epithelial microchannels allows for stromal Rf concentrations high enough to perform true transepithelial crosslinking.

Femtosecond laser ablation of the stapes.

A femtosecond laser, normally used for LASIK eye surgery, is used to perforate cadaveric human stapes. The thermal side effects of bone ablation are measured with a thermocouple in an inner ear model and are found to be within acceptable limits for inner ear surgery. Stress and acoustic events, recorded with piezoelectric film and a microphone, respectively, are found to be negligible. Optical microscopy, scanning electron microscopy, and optical coherence tomography are used to confirm the precision of the ablation craters and lack of damage to the surrounding tissue. Ablation is compared to that from an Er:YAG laser, the current laser of choice for stapedotomy, and is found to be superior. Ultra-short-pulsed lasers offer a precise and efficient ablation of the stapes, with minimal thermal and negligible mechanical and acoustic damage. They are, therefore, ideal for stapedotomy operations.

Nonlinear Optical Corneal Crosslinking, Mechanical Stiffening, and Corneal Flattening Using Amplified Femtosecond Pulses.

PURPOSE: We have shown that nonlinear optical corneal crosslinking (NLO CXL) and stiffening can be achieved in ex vivo rabbit corneas using an 80-MHz, 760-nm femtosecond (FS) laser, however the required power was beyond the American National Standard Institute limit. The purpose of this study was to test the efficacy of amplified FS pulses to perform CXL to reduce power by increasing pulse energy. METHODS: A variable numerical aperture laser scanning delivery system was coupled to a 1030-nm laser with a noncollinear optical parametric amplifier to generate 760 nm, 50 to 150 kHz amplified FS pulses with 79.5-µm axial and 2.9-µm lateral two-photon focal volume. Ex vivo rabbit corneas received NLO CXL, and effectiveness was assessed by measuring collagen autofluorescence (CAF) and mechanical stiffening. NLO CXL was also performed in 14 live rabbits, and changes in corneal topography were measured using an Orbscan. RESULTS: Amplified pulses (0.3 µJ) generated significant CAF that increased logarithmically with decreasing scan speed; achieving equivalent CAF to UVA CXL at 15.5 mm/s. Indentation testing detected a 62% increase in stiffness compared to control, and corneal topography measurements revealed a significant decrease of 1.0 ± 0.8 diopter by 1 month (P < 0.05). CONCLUSIONS: These results show that NLO CXL using amplified pulses can produce corneal collagen CXL comparable to UVA CXL. TRANSLATIONAL RELEVANCE: NLO CXL using amplified pulses can produce corneal CXL comparable to UVA CXL, suggesting a potential clinical application in which NLO CXL can be used to perform personalized crosslinking for treatment of refractive errors and keratoconus.

Characterization of submicrojoule femtosecond laser corneal tissue dissection.

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 microm flap thickness, 9.0 mm flap diameter, 60-degree hinge length, 65-degree side cut, 0.4 muJ or 0.7 muJ raster energy, 7 microm x 7 microm or 9 microm x 9 microm spot/line separation, and 1 muJ 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.

Evolution of the vertebrate corneal stroma.

Although the cornea is the major refractive element of the eye, the mechanisms controlling corneal shape and hence visual acuity remain unknown. To begin to address this question we have used multiphoton, non-linear optical microscopy to image second harmonic generated signals (SHG) from collagen to characterize the evolutionary and structural changes that occur in the collagen architecture of the corneal stroma. Our studies show that there is a progression in complexity of the stromal collagen organization from lower (fish and amphibians) to higher (birds and mammals) vertebrates, leading to increasing tissue stiffness that may control shape. In boney and cartilaginous fish, the cornea is composed of orthogonally arranged, rotating collagen sheets that extend from limbus to limbus with little or no interaction between adjacent sheets, a structural paradigm analogous to 'plywood'. In amphibians and reptiles, these sheets are broken down into broader lamellae that begin to show branching and anastomosing with adjacent lamellae, albeit maintaining their orthogonal, rotational organization. This paradigm is most complex in birds, which show the highest degree of lamellar branching and anastomosing, forming a 'chicken wire' like pattern most prominent in the midstroma. Mammals, on the other hand, diverged from the orthogonal, rotational organization and developed a random lamellar pattern with branching and anastomosing appearing highest in the anterior stroma, associated with higher mechanical stiffness compared to the posterior stroma.