Bryan E. Jester

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, Bowmans 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.

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 &mgr;m). 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 × 466 × 150 &mgr;m) were taken, covering the entire tissue (15 mm × 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 &mgr;m lateral, 2.0 &mgr;m 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 (Bowmans layer). Conclusions: We conclude that HRMac using NLO-based tomography provides a powerful new tool to assess collagen structural organization within the cornea.

Volumetric reconstruction of the mouse meibomian gland using high-resolution nonlinear optical imaging.

Recent studies suggest that mouse meibomian glands (MG) undergo age‐related atrophy that mimics changes seen in age‐related human MG dysfunction (MGD). To better understand the structural/functional changes that occur during aging, this study developed an imaging approach to generate quantifiable volumetric reconstructions of the mouse MG and measure total gland, cell, and lipid volume. Mouse eyelids were fixed in 4% paraformaldehyde, embedded in LR White resin and serially sectioned. Sections were then scanned using a 20× objective and a series of tiled images (1.35 × 1.35 × 0.5 mm) with a pixel size of 0.44 μm lateral and 2 μm axial were collected using a Zeiss 510 Meta LSM and a femtosecond laser to simultaneously detect second harmonic generated (SHG) and two‐photon excited fluorescence (TPEF) signals from the tissue sections. The SHG signal from collagen was used to outline and generate an MG mask to create surface renderings of the total gland and extract relevant MG TPEF signals that were later separated into the cellular and lipid compartments. Using this technique, three‐dimensional reconstructions of the mouse MG were obtained and the total, cell, and lipid volume of the MG measured. Volumetric reconstructions of mouse MG showed loss of acini in old mice that were not detected by routine histology. Furthermore, older mouse MG had reduced total gland volume that is primarily associated with loss of the lipid volume. These findings suggest that mice MG undergo “dropout” of acini, similar to that which occurs in human age‐related MGD. Anat Rec, 2011.

In vivo non-linear optical (NLO) imaging in live rabbit eyes using the Heidelberg Two-Photon Laser Ophthalmoscope.

Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW fs laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 mum diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 h after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas.

High resolution macroscopy (HRMac) of the eye using nonlinear optical imaging

Non-linear optical (NLO) imaging using femtosecond lasers provides a non-invasive means of imaging the structural organization of the eye through the generation of second harmonic signals (SHG). While NLO imaging is able to detect collagen, the small field of view (FoV) limits the ability to study how collagen is structurally organized throughout the larger tissue. To address this issue we have used computed tomography on optical and mechanical sectioned tissue to greatly expand the FoV and provide high resolution macroscopic (HRMac) images that cover the entire tissue (cornea and optic nerve head). Whole, fixed cornea (13 mm diameter) or optic nerve (3 mm diameter) were excised and either 1) embedded in agar and sectioned using a vibratome (200-300 um), or 2) embedded in LR White plastic resin and serially sectioned (2 um). Vibratome and plastic sections were then imaged using a Zeiss LSM 510 Meta and Chameleon femtosecond laser to generate NLO signals and assemble large macroscopic 3-dimensional tomographs with high resolution that varied in size from 9 to 90 Meg pixels per plane having a resolution of 0.88 um lateral and 2.0 um axial. 3-D reconstructions allowed for regional measurements within the cornea and optic nerve to quantify collagen content, orientation and organization over the entire tissue. We conclude that NLO based tomography to generate HRMac images provides a powerful new tool to assess collagen structural organization. Biomechanical testing combined with NLO tomography may provide new insights into the relationship between the extracellular matrix and tissue mechanics.

Indentation Testing of the Optic Nerve Head and Posterior Sclera

The mechanical behavior of the optic nerve head (ONH) and the surrounding sclera play important roles in the development of optic neuropathy. We assessed the indentation behavior of the ONH and the surrounding sclera in unfixed human autopsy eyes from individuals between 27 and 87 years of age. Our testing device utilized a round-tipped 250 µm diameter stainless steel probe to measure the force applied in discrete 50 µm steps from the surface of the tissues to a depth of 400 µm. Thirteen eyes from eight individuals were indented in various locations within and around the ONH and the posterior sclera. Tissue thickness, force and depth were recorded at each position tested. Data was analyzed by evaluating the recorded force as a function of the depth of indentation. The data showed that, for both the sclera and the ONH, the force rose exponentially with increased depth of indentation. Apparent stiffness values were estimated using 2 different equations that assess soft biologic tissues. Results showed that significantly higher levels of force were required with aging to indent the posterior sclera, with a corresponding increase in stiffness values. However, no significant increase in indentation force as a function of age was noted for the ONH. Interestingly, the data suggests that some individuals have relatively large differences in the stiffness between the ONH and the posterior sclera, while others show less difference regardless of age. This data supports the notion that some individuals have relatively softer ONH tissue, and as the sclera becomes stiffer with age, this difference is magnified. This difference may be important in understanding the biomechanical contribution to the individually different susceptibility to glaucoma.