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Study: Mapping lamellar structure of human corneas by use of polarization-resolved-SHG

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The study of pathological tissues will enable to establish the role of corneal structure in certain diseases.

(Image Credit: AdobeStock)

(Image Credit: AdobeStock)

A key role of the cornea is to aid in the focusing of light on the retina. With a curved shape and ability to focus light, it has to be stable to provide a sharp image amid myriad shocks and rubs that occur during the course of a day.

The cornea is made up of several hundred collagen lamellae stacked one on top of the other with various in-plane orientations, but the transparency of this tissue, essential for visual acuity, is an obstacle to optical imaging of its structure using conventional optical microscopes. As a result, poor characterization of the lamellar structure of the cornea today limits researchers’ understanding of the link between structure and function of this tissue, particularly in terms of its mechanical properties. Another limitation concerns the understanding of certain pathologies linked to a defective structure, such as keratoconus.1

In a new paper1 published in Light Science & Application, a team of scientists, led by Professor Marie-Claire Schanne-Klein from the Laboratory for Optics and Biosciences at Ecole Polytechnique, CNRS, Inserm and Institut Polytechnique de Paris, Palaiseau, France, have used a recent three-dimensional optical imaging technique: second harmonic generation (SHG) microscopy, which enables specific visualization of collagen without any prior labeling.

According to the study, the originality of researchers’ approach lies in playing on the polarization of light to reveal the direction of the collagen fibrils that compose the lamellae of the cornea. This imaging is performed in reflection, using epi-detection and may be further developed to eventually enable in vivo diagnosis.1

Moreover, the researchers noted in the study that a key advantage of this configuration is that it provides far more accurate results than transmission. The reason for this, according to the researchers, is the coherence length of SHG processes is shorter in the backward direction than in the forward direction, which in practice ensures better spatial resolution.

The researchers characterized 10 human corneas, according to the study, and a careful validation was performed by two self-referenced methods, as SHG polarimetry had never been implemented on tissue of such thickness (around 600 µm).1 In particular, the researchers have verified that the same structure is obtained by imaging the cornea in two different orientations, according to the study, which has also shown that for the first time that, while the lamellae of the human cornea are globally oriented in two perpendicular directions, their main direction changes with depth.

A simplified scheme of the polarization-resolved SHG microscope with epi-detection. The polarization is controlled by two waveplates at the back aperture of the objective lens. b, an example of a polarimetric diagram obtained in every voxel of the cornea. The double-arrow indicates the orientation of collagen in the imaging plane. c, Three-dimensional reconstruction of the lamellar structure of a human cornea. The colors indicate the direction of the collagen lamellae in the imaging plane, as indicated in the color wheel insert. Image size is 250 ×250×600 µm³. The anterior part of the cornea is at the top of the image. (Image courtesy of Marie-Claire/SCHANNE-KLEIN)

A simplified scheme of the polarization-resolved SHG microscope with epi-detection. The polarization is controlled by two waveplates at the back aperture of the objective lens. b, an example of a polarimetric diagram obtained in every voxel of the cornea. The double-arrow indicates the orientation of collagen in the imaging plane. c, Three-dimensional reconstruction of the lamellar structure of a human cornea. The colors indicate the direction of the collagen lamellae in the imaging plane, as indicated in the color wheel insert. Image size is 250 ×250×600 µm³. The anterior part of the cornea is at the top of the image. (Image courtesy of Marie-Claire/SCHANNE-KLEIN)

The study demonstrates potential new characterizations of the cornea, including mapping the size and distribution of lamellae as a function of depth, but also as a function of position (center versus periphery of the tissue). According to the study, the information will feed into mechanical modelling of corneal behavior in response to variations in intraocular pressure or healing processes. Finally, the study of pathological tissues will enable to establish the role of corneal structure in certain diseases.1

Other innovations

More recently, innovations addressing corneal diseases have proliferated. In particular, Fuchs endothelial corneal disease, keratoconus, bullous keratopathy, and other corneal dystrophies have sparked the development of many new technologies.

More recently, innovations addressing corneal diseases have proliferated. In particular, Fuchs endothelial corneal disease, keratoconus, bullous keratopathy, and other corneal dystrophies have sparked the development of many new technologies.2

Clara C. Chan, MD, FRCSC, FACS, pointed out that cell therapy involving fully differentiated HCECs holds the most immediate promise for patients suffering from corneal dystrophies and may potentially address multiple drawbacks of current standards of care. In addition to addressing the chronic worldwide shortage of donor corneas, cell therapy procedures may prove to be less invasive and more tolerable than endothelial keratoplasties due to lower risk of rejection vs EK or penetrating keratoplasty, which in turn could mean less onerous recovery for patients.

“It’s an exciting time for innovations targeting corneal dystrophies and a space for us all to follow with interest,” Chan said.

References
  1. Raoux, C., Chessel, A., Mahou, P. et al. Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy. Light Sci Appl 12, 190 (2023). https://doi.org/10.1038/s41377-023-01224-0
  2. Orash Mahmoud Salehi A, Keshel SH, Sefat F, Tayebi L. Use of polycaprolactone in corneal tissue engineering: a review. Mater Today Commun. 2021;27(6):102402. doi:10.1016/j.mtcomm.2021.102402
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