Human graft cornea and laser incisions imaging with micrometer scale resolution full-field optical coherence tomography

Latour, Gaël; Georges, Gaëlle; Lamoine, Laure Siozade; Deumié, Carole; Conrath, J.; Hoffart, L.

doi:10.1117/1.3486544

Cited by 18 publications

(12 citation statements)

References 49 publications

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“…Commercially available techniques, such as confocal reflectance microscopy [1] or optical coherence tomography (OCT) [2,3], enable three-dimensional (3D) cell-scale imaging of cornea. However, these techniques lack specificity or contrast when looking at the collagen organization of the corneal stroma.…”

Section: Introductionmentioning

confidence: 99%

In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy

Latour¹,

Gusachenko²,

Kowalczuk³

et al. 2011

Biomed. Opt. Express

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The transparency and mechanical strength of the cornea are related to the highly organized three-dimensional distribution of collagen fibrils. It is of great interest to develop specific and contrasted in vivo imaging tools to probe these collagenous structures, which is not available yet. Second Harmonic Generation (SHG) microscopy is a unique tool to reveal fibrillar collagen within unstained tissues, but backward SHG images of cornea fail to reveal any spatial features due to the nanometric diameter of stromal collagen fibrils. To overcome this limitation, we performed polarization-resolved SHG imaging, which is highly sensitive to the sub-micrometer distribution of anisotropic structures. Using advanced data processing, we successfully retrieved the orientation of the collagenous fibrils at each depth of human corneas, even in backward SHG homogenous images. Quantitative information was also obtained about the submicrometer heterogeneities of the fibrillar collagen distribution by measuring the SHG anisotropy. All these results were consistent with numerical simulation of the polarization-resolved SHG response of cornea. Finally, we performed in vivo SHG imaging of rat corneas and achieved structural imaging of corneal stroma without any labeling. Epi-detected polarization-resolved SHG imaging should extend to other organs and become a new diagnosis tool for collagen remodeling.

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Section: Introductionmentioning

confidence: 99%

In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy

Latour¹,

Gusachenko²,

Kowalczuk³

et al. 2011

Biomed. Opt. Express

Self Cite

124

101

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show abstract

“…Most of the studies dedicated to the study of the scattering properties of the cornea focuse on the transmitted half-space, showing an increase of the scattering with the thickness 12 performed also measurements in the reflected half-space 13 . Indeed, we aim to propose such characterization before removal of the cornea on donor eyes, or by extension to in-vivo studies.…”

Section: Resultsmentioning

confidence: 99%

Scattering properties and transparency characterization of human corneal grafts

et al. 2011

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The cornea is the single human tissue being transparent. This unique property may be explained by the particular structure of the cornea, but the precise role of each of its constituents remains unsolved. On other matter, prior to corneal transplant, graft must be evaluated during a sorting procedure where a technician assesses of its transparency quality. Nevertheless, this criterion remains subjective and qualitative. This study proposes to combine 3D imagery using Full-Field Optical Coherence Tomography jointly with angular resolved scattering measurement to achieve a quantitative transparency characterization of the cornea. The OCT provides micrometric resolution structural information about the cornea, and we observe the evolution occurring when oedema develops within the tissue. Scattering properties are evaluated and compared parallely, as the transparency of the graft. A close link between the scattering intensity level of the cornea and its thickness is highlighted through this study. Furthermore, the three-dimensional imagery offers a view over the structural modifications leading to a change in transparency, and the combination with scattering properties measurement provides clues over the characteristic scale of scatterers to consider for a better understanding of corneal transparency evolution. Achieving an objective and quantified parameter for the transparency would be helpful for a more efficient corneal graft sorting, and may be able to detect the presence of localized wounds as the ones related to a previous refractive surgery. However, the study of graft nearly eligible for corneal transplant would be needed to confirm the results this study presents. CONTEXT AND PROBLEMATICLight transmission inside the human ocular globe is ensured by a disc with a mean diameter of 11 mm and an average thickness of 600 µm: the cornea. It is the outermost layer of the eye, and the single human tissue with the lens being transparent. It offers an averaged transmission factor superior to 90% in the visible spectral range (400 to 800 nm) 1 , several considerations being involved in such phenomenon.This transparency is firstly due to the avascularization of the cornea, the oxygen and nutriments supply being supported by tears and aqueous. Because of the absence of haemoglobin, the light transmission is not only restricted to the "therapeutic window" (wavelengths from 650 nm to 1 µm) usually available in biological tissues. Nevertheless, the main explanation of this transparency property is linked to the specific and ordered structure of the cornea. The cornea consists in the superimposition of 5 distinct layers, the most important being the stroma which represents 90% of the whole thickness. It is composed of collagen fibrils with a diameter of 20 to 35 nm, disposed in parallel the one to the others with a 40 nm space, within 2 µm thick lamellas 2 . These lamellas are piled up all along the thickness of the cornea with slight angles between them. Several theories were proposed implying the highly ordered structure ...

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“…Note that Optical Coherence Tomography (OCT), that is another well-established optical technique in ophthalmology [24], exhibits the same drawbacks. While optical sectioning is performed in a different way as for CM, by use of interferometry, this imaging technique is based on the same physical contrast as CM and the observed structures are intrinsically similar [25], [26]. Thus, neither better contrast nor better specificity is expected by using OCT instead of CM.…”

Section: Discussionmentioning

confidence: 99%

Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The Potential of Second Harmonic Generation Microscopy

et al. 2012

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BackgroundSecond Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet’s membrane, in the posterior cornea.Methodology/Principal FindingsWe studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet’s membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats.Conclusions/SignificanceOur study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.

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Human graft cornea and laser incisions imaging with micrometer scale resolution full-field optical coherence tomography

Cited by 18 publications

References 49 publications

In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy

In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy

Scattering properties and transparency characterization of human corneal grafts

Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The Potential of Second Harmonic Generation Microscopy

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