Custom built nonlinear optical crosslinking (NLO CXL) device capable of producing mechanical stiffening in ex vivo rabbit corneas.

The purpose of this study was to develop and test a nonlinear optical device to photoactivate riboflavin to produce spatially controlled collagen crosslinking and mechanical stiffening within the cornea. A nonlinear optical device using a variable numerical aperture objective was built and coupled to a Chameleon femtosecond laser. Ex vivo rabbit eyes were then saturated with riboflavin and scanned with various scanning parameters over a 4 mm area in the central cornea. Effectiveness of NLO CXL was assessed by evaluating corneal collagen auto fluorescence (CAF). To determine mechanical stiffening effects, corneas were removed from the eye and subjected to indentation testing using a 1 mm diameter probe and force transducer. NLO CXL was also compared to standard UVA CXL. The NLO CXL delivery device was able to induce a significant increase in corneal stiffness, comparable to the increase produced by standard UVA CXL.

Measurement of corneal elasticity with an acoustic radiation force elasticity microscope.

To investigate the role of collagen structure in corneal biomechanics, measurement of localized corneal elasticity with minimal destruction to the tissue is necessary. We adopted the recently developed acoustic radiation force elastic microscopy (ARFEM) technique to measure localize biomechanical properties of the human cornea. In ARFEM, a low-frequency, high-intensity acoustic force is used to displace a femtosecond laser-generated microbubble, while high-frequency, low-intensity ultrasound is used to monitor the position of the microbubble within the cornea. Two ex vivo human corneas from a single donor were dehydrated to physiologic thickness, embedded in gelatin and then evaluated using the ARFEM technique. In the direction perpendicular to the corneal surface, ARFEM measurements provided elasticity values of E = 1.39 ± 0.28 kPa for the central anterior cornea and E = 0.71 ± 0.21 kPa for the central posterior cornea in pilot studies. The increased value of corneal elasticity in the anterior cornea correlates with the higher density of interweaving lamellae in this region.

Nonlinear optical collagen cross-linking and mechanical stiffening: a possible photodynamic therapeutic approach to treating corneal ectasia.

In this study we test the hypothesis that nonlinear optical (NLO) multiphoton photoactivation of riboflavin using a focused femtosecond (FS) laser light can be used to induce cross-linking (CXL) and mechanically stiffen collagen as a potential clinical therapy for the treatment of keratoconus and corneal ectasia. Riboflavin-soaked, compressed collagen hydrogels are cross-linked using a FS laser tuned to 760 nm and set to either 100 mW (NLO CXL I) or 150 mW (NLO CXL II) of laser power. FS pulses are focused into the hydrogel using a 0.75 NA objective lens, and the hydrogel is three-dimensionally scanned. Measurement of hydrogel stiffness by indentation testing show that the calculated elastic modulus (E) values are significantly increased over twofold following NLO CXL I and II compared with baseline values (P<0.05). Additionally, no significant differences are detected between NLO CXL and single photon, UVA CXL (P>0.05). This data suggests that NLO CXL has a comparable effect to conventional UVA CXL in mechanically stiffening collagen and may provide a safe and effective approach to localize CXL at different regions and depths within the cornea.

Nonlinear optical macroscopic assessment of 3-D corneal collagen organization and axial biomechanics.

PURPOSE: To characterize and quantify the collagen fiber (lamellar) organization of human corneas in three dimensions by using nonlinear optical high-resolution macroscopy (NLO-HRMac) and to correlate these findings with mechanical data obtained by indentation testing of corneal flaps. METHODS: Twelve corneas from 10 donors were studied. Vibratome sections, 200 µm thick, from five donor eyes were cut along the vertical meridian from limbus to limbus (arc length, 12 mm). Backscattered second harmonic-generated (SHG) NLO signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 3-D mosaic (pixel resolution: 0.44 µm lateral, 2 µm axial). Collagen fiber intertwining was quantified by determining branching point density as a function of stromal depth. Mechanical testing was performed on corneal flaps from seven additional eyes. Corneas were cut into three layers (anterior, middle, and posterior) using a femtosecond surgical laser system and underwent indentation testing to determine the elastic modulus for each layer. RESULTS: The 3-D reconstructions revealed complex collagen fiber branching patterns in the anterior cornea, with fibers extending from the anterior limiting lamina (ALL, Bowman's layer), intertwining with deeper fibers and reinserting back to the ALL, forming bow spring-like structures. Measured branching-point density was four times higher in the anterior third of the cornea than in the posterior third and decreased logarithmically with increasing distance from the ALL. Indentation testing showed an eightfold increase in elastic modulus in the anterior stroma. CONCLUSIONS: The axial gradient in lamellar intertwining appears to be associated with an axial gradient in the effective elastic modulus of the cornea, suggesting that collagen fiber intertwining and formation of bow spring-like structures provide structural support similar to cross-beams in bridges and large-scale structures. Future studies are necessary to determine the role of radial and axial structural-mechanical heterogeneity in controlling corneal shape and in the development of keratoconus, astigmatism, and other refractive errors.

Quantitative assessment of UVA-riboflavin corneal cross-linking using nonlinear optical microscopy.

PURPOSE: Corneal collagen cross-linking (CXL) by the use of riboflavin and ultraviolet-A light (UVA) is a promising and novel treatment for keratoconus and other ectatic disorders. Since CXL results in enhanced corneal stiffness, this study tested the hypothesis that CXL-induced stiffening would be proportional to the collagen autofluorescence intensity measured with nonlinear optical (NLO) microscopy. METHODS: Rabbit eyes (n = 50) were separated into five groups including: (1) epithelium intact; (2) epithelium removed; (3) epithelium removed and soaked in riboflavin, (4) epithelium removed and soaked in riboflavin, with 15 minutes of UVA exposure; and (5) epithelium removed and soaked in riboflavin, with 30 minutes of UVA exposure. Corneal stiffness was quantified by measuring the force required to displace the cornea 500 µm. Corneas were then fixed in paraformaldehyde and sectioned, and the collagen autofluorescence over the 400- to 450-nm spectrum was recorded. RESULTS: There was no significant difference in corneal stiffness among the three control groups. Corneal stiffness was significantly and dose dependently increased after UVA (P < 0.0005). Autofluorescence was detected only within the anterior stroma of the UVA-treated groups, with no significant difference in the depth of autofluorescence between different UVA exposure levels. The signal intensity was also significantly increased with longer UVA exposure (P < 0.001). Comparing corneal stiffness with autofluorescence intensity revealed a significant correlation between these values (R(2) = 0.654; P < 0.0001). CONCLUSIONS: The results of this study indicate a significant correlation between corneal stiffening and the intensity of collagen autofluorescence after CXL. This finding suggests that the efficacy of CXL in patients could be monitored by assessing collagen autofluorescence.

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.

Evaluating corneal collagen organization using high-resolution nonlinear optical macroscopy.

PURPOSE: Recent developments in nonlinear optical (NLO) imaging using femtosecond lasers provides a noninvasive method for detecting collagen fibers by imaging second harmonic-generated (SHG) signals. However, this technique is limited by the small field of view necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the field of view to assess the entire collagen structure using high-resolution macroscopic (HRMac) imaging. METHODS: Intact human eyes were fixed under pressure, and the whole cornea (13-mm diameter) was excised and embedded in low-melting point agar for vibratome sectioning (200-300 microm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser (Carl Zeiss Microimaging Inc., Thornwood, NY) to generate SHG images. For each vibratome section, an overlapping series of three-dimensional data sets (466 x 466 x 150 microm) were taken, covering the entire tissue (15 mm x 6 mm area) using a motorized, mechanical stage. The three-dimensional data sets were then concatenated to generate an NLO-based tomograph. RESULTS: The HRMac of the cornea yielded large macroscopic (80 megapixels per plane), three-dimensional tomographs with high resolution (0.81 microm lateral, 2.0 microm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented, and extracted. Three-dimensional reconstructions suggested that the anterior cornea comprises highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's layer). CONCLUSIONS: We conclude that HRMac using NLO-based tomography provides a powerful new tool to assess collagen structural organization within the cornea.

3D finite element model of aqueous outflow to predict the effect of femtosecond laser created partial thickness drainage channels.

BACKGROUND AND OBJECTIVES: Partial thickness drainage channels can be created with femtosecond lasers in the translucent sclera for the potential treatment of glaucoma. We present a 3D finite element model (FEM) that can predict the effect of these channels on aqueous humor (AH) outflow and intraocular pressure (IOP). STUDY DESIGN/MATERIALS AND METHODS: A 3D model was developed based on a 2D model for the intact eye using COMSOL (Comsol, Inc., MA) finite element software. Different values of permeability were entered into the 3D model for the AH pathway and for the partial thickness channel. To obtain experimental data for model validation, one partial thickness channel was created in each of three enucleated rabbit eyes with a femtosecond laser tuned to 1.7 microm wavelength. Aqueous outflow rates were measured with the perfusion method before and after the laser treatments at different levels of IOP and then compared to IOP values predicted by the model. RESULTS: The experiments indicated that the rate of the AH outflow was increased in each of three eyes after the laser treatment. Assuming a constant rate of AH production the 3D model predicted IOP reductions ranging from 67.2% to 80.6% as the effect of the laser created channels. These predictions were in reasonable agreement with experimentally adjusted IOP values during the perfusion measurements. CONCLUSIONS: The developed 3D FEM has the potential to predict IOP reduction caused by partial thickness drainage channels created with the femtosecond laser in the sclera. Such a model may also be used to determine optimal channel dimensions for a specified increase in outflow facility and reduction in IOP.