Analysis of spatial lamellar distribution from adaptive-optics second harmonic generation corneal images (original) (raw)
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Journal of biophotonics, 2016
Keratoconus is an eye disorder that causes the cornea to take an abnormal conical shape, thus impairing its refractive functions and causing blindness. The late diagnosis of keratoconus is among the principal reasons for corneal surgical transplantation. This pathology is characterized by a reduced corneal stiffness in the region immediately below Bowman's membrane, probably due to a different lamellar organization, as suggested by previous studies. Here, the lamellar organization in this corneal region is characterized in three dimensions by means of second-harmonic generation (SHG) microscopy. In particular, a method based on a three-dimensional correlation analysis allows to probe the orientation of sutural lamellae close to the Bowman's membrane, finding statistical differences between healthy and keratoconic samples. This method is demonstrated also in combination with an epi-detection scheme, paving the way for a potential clinical ophthalmic application of SHG microsc...
Biophysical Journal, 2009
The purpose of this study was to image and quantify the structural changes of corneal edema by second harmonic generation (SHG) microscopy. Bovine cornea was used as an experimental model to characterize structural alterations in edematous corneas. Forward SHG and backward SHG signals were simultaneously collected from normal and edematous bovine corneas to reveal the morphological differences between them. In edematous cornea, both an uneven expansion in the lamellar interspacing and an increased lamellar thickness in the posterior stroma (depth > 200 mm) were identified, whereas the anterior stroma, composed of interwoven collagen architecture, remained unaffected. Our findings of heterogeneous structural alteration at the microscopic scale in edematous corneas suggest that the strength of collagen cross-linking is heterogeneous in the corneal stroma. In addition, we found that qualitative backward SHG collagen fiber imaging and depth-dependent signal decay can be used to detect and diagnose corneal edema. Our work demonstrates that SHG imaging can provide morphological information for the investigation of corneal edema biophysics, and may be applied in the evaluation of advancing corneal edema in vivo.
Photonics
In this paper, a Second-Harmonic-Generation (SHG) microscope was used to study the relationship between collagen structural arrangement, image quality and polarization sensitivity in human corneas with different organizations. The degree of order (or alternatively, the Structural Dispersion, SD) was quantified using the structure tensor method. SHG image quality was evaluated with different objective metrics. Dependence with polarization was quantified by means of a parameter defined as polarimetric modulation, which employs polarimetric SHG images acquired with four independent polarization states. There is a significant exponential relationship between the quality of the SHG images and the SD of the samples. Moreover, polarization sensitivity strongly depends on collagen arrangement. For quasi- or partially organized specimens, there is a polarization state that noticeably improves the image quality, providing additional information often not seen in other SHG images. This does no...
Evaluating Corneal Collagen Organization Using High-Resolution Nonlinear Optical Macroscopy
Eye & Contact Lens: Science & Clinical Practice, 2010
Purpose-Recent developments in non-linear optical (NLO) imaging using femtosecond lasers provides a non-invasive method for detecting collagen fibers by imaging second harmonic generated (SHG) signals. However, this technique is limited by the small field of view (FoV) necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the FoV in order 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) excised and embedded in low melting point agar for vibratome sectioning (200-300 μm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser to generate SHG images. For each vibratome section, an overlapping series of 3-D data sets (466 × 466 × 150 μm) were taken covering the entire tissue (15 mm × 6 mm area) using a motorized, mechanical stage. The 3-D data sets were then concatenated to generate an NLO based tomograph. Results-HRMac of the cornea yielded large macroscopic (80 Meg Pixels per plane), 3dimensional tomographs with high resolution (0.81 μm later, 2.0 μm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented and extracted. 3-D reconstructions suggested that the anterior cornea is comprised of highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's Layer). Conclusion-We conclude that HRMac using NLO based tomography provides a powerful new tool to assess collagen structural organization within the cornea.
Nonlinear Optical Macroscopic Assessment of 3-D Corneal Collagen Organization and Axial Biomechanics
Investigative Opthalmology & Visual Science, 2011
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 m 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 m lateral, 2 m 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. (In