Quantitative Analysis of the Corneal Collagen Distribution after<i> In Vivo</i> Cross-Linking with Second Harmonic Microscopy

Bueno, Juan M.; Ávila, Francisco J.; Martínez‐García, Carmen

doi:10.1155/2019/3860498

Cited by 21 publications

(16 citation statements)

References 56 publications

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“…The structure tensor used here has been reported to be useful to compare the spatial distribution of structural features in pairs of 2P images 45,46 . In addition, another comparison using the Mean Image Gradient (MIG) has also been calculated.…”

Section: Resultsmentioning

confidence: 99%

In vivo two-photon microscopy of the human eye

Ávila

Gambin

Artal

et al. 2019

Sci Rep

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Two-photon (2P) microscopy is a powerful tool for imaging and exploring label-free biological tissues at high resolution. Although this type of microscopy has been demonstrated in ex vivo ocular tissues of both humans and animal models, imaging the human eye in vivo has always been challenging. This work presents a novel compact 2P microscope for non-contact imaging of the anterior part of the living human eye. The performance of the instrument was tested and the maximum permissible exposure to protect ocular tissues established. To the best of our knowledge, 2P images of the in vivo human cornea, the sclera and the trabecular meshwork are shown for the very first time. Acquired images are of enough quality to visualize collagen arrangement and morphological features of clinical interest. Future implementations of this technique may constitute a potential tool for early diagnosis of ocular diseases at submicron scale.

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Section: Resultsmentioning

confidence: 99%

In vivo two-photon microscopy of the human eye

Ávila

Gambin

Artal

et al. 2019

Sci Rep

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“…SHG imaging offers inherent confocality and provides depth-resolved information in both healthy and pathological corneas. In particular, the ST has recently been shown to be very sensitive to changes in fiber orientation with depth in postsurgery (cross-linked) corneas 44…”

Section: Discussionmentioning

confidence: 99%

Quantitative Discrimination of Healthy and Diseased Corneas With Second Harmonic Generation Microscopy

Ávila

Artal

Bueno

2019

Trans. Vis. Sci. Tech.

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Purpose: To analyze the spatial organization of pathological corneas with second harmonic generation (SHG) imaging and to provide a proof of concept to objectively distinguish these from the healthy corneas. Methods: A custom-built SHG microscope was used to image the anterior stroma of ex vivo corneas, both control and affected by some representative pathologies. The structure tensor (ST) was employed as a metric to explore and quantify the alterations in the spatial distribution of the collagen lamellae. Results: The collagen arrangement differed between healthy and pathological samples. The former showed a regular distribution and a low structural dispersion (SD , 408) within the stroma with a well-defined dominant orientation. This regular arrangement drastically turns into a disorganized pattern in pathological corneas (SD. 408). Conclusions: The combination of SHG imaging and the ST allows obtaining quantitative information to differentiate the stromal collagen organization in healthy and diseased corneas. This approach represents a feasible and powerful technique with potential applications in clinical corneal diagnoses. Translational Relevance: The ST applied to SHG microscopy images of the corneal stroma provides an experimental objective score to differentiate control from pathological or damaged corneas. Future implementations of this technique in clinical environments might might be a promising tool in Ophthalmology, not only to diagnose and monitor corneal diseases, but also to follow-up surgical outcome.

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“…In order to study corneal structure, a variety of imaging techniques such as optical microscopy, second harmonic generation (SHG) imaging, electron microscopy and X-ray scattering have been used to study normal and abnormal samples in humans and other species [11][12][13][14][15][16]. For example, SHG imaging showed that cross-linking did not significantly affect structures of corneas with well-organized lamellae [17]. Moreover, optical and electron microscopies were used to study corneal development in the chick model and it was found that rotation of collagen lamellae evolves with maturation.…”

Section: Introductionmentioning

confidence: 99%

Second harmonic generation imaging reveals asymmetry in the rotational helicity of collagen lamellae in chicken corneas

Lee

Chen

Dong

2019

Biomed. Opt. Express

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High tensile strength and optical clarity are unique properties of the cornea. These features are dictated by the three-dimensional architecture of corneal lamellae. Therefore, understanding the microscopic details of the cornea's structural organization may contribute to the development of artificial cornea for the treatment of corneal diseases. In this study, the combination of forward second harmonic generation (SHG) microcopy and fast Fourier-transform based image analysis was used to characterize the depth-dependent superstructure of chicken corneal stroma. Our results show that from the surface, adjacent lamellae of anterior chicken cornea lamella rotate in a counterclockwise direction, and the same rotational helicity is observed in left and right corneas. Furthermore, the overall average rotational pitch of lamellae is 0.92 ± 0.11 degree/µm which persists for 176 ± 14 µm in the anterior stroma. As depth further increased, the rate of lamellar rotation decreases. Upon reaching posterior stroma, lamellar orientation remains constant. Throughout the stroma, collagen lamellae in chicken rotate a total of 169 ± 21 degrees. The lack of lamellar rotation in posterior stroma suggests that packing efficiency cannot be used to explain the helicity of depth-dependent rotation of anterior stroma. In addition, although the right cornea has a higher rotational pitch (0.95 ± 11 vs 0.90 ± 10 degrees/µm) and thinner anterior stroma (173 ± 13 vs 179 ± 14 µm) than the left cornea, the two effects cancel each other out and result in similar total angular rotation of anterior stroma (161 ± 23 and 165 degrees ± 21). Finally, our observation of a total angular rotation of 169 ± 21 degrees shows that within experimental error, chicken cornea lamellae rotate around 180 degrees or half of a complete turn. Additional studies are needed to arrive at an explanation of chicken superstructure in three dimensions.

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Quantitative Analysis of the Corneal Collagen Distribution after In Vivo Cross-Linking with Second Harmonic Microscopy

Cited by 21 publications

References 56 publications

In vivo two-photon microscopy of the human eye

In vivo two-photon microscopy of the human eye

Quantitative Discrimination of Healthy and Diseased Corneas With Second Harmonic Generation Microscopy

Second harmonic generation imaging reveals asymmetry in the rotational helicity of collagen lamellae in chicken corneas

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