Polarimetric interferometry to objectively evaluate the optical properties of corneal stroma

A new non-invasive method, based on the interferometric analysis of diff ractive and polarizing eff ects related to the birefringent properties of corneal collagen fibrils, has been developed to objectively evaluate the optical properties of the stroma. The new method shows a relevant impact on corneal surgeries specifically for lamellar transplantation where, due to the polarizing properties of the stroma, the alignment between collagen fibrils of donor corneas with patient collagen fibril orientation has shown an improvement of visual acuity postoperatively. Further studies on the regularity of the corneal isogyre pattern are showing this new method has a strong impact in early-stage diagnosis of corneal disease.


Introduction
The cornea has a dual function, optical and mechanical.The human cornea provides two-thirds of the refractive power of the eye and the requisite durability to maintain its shape, notwithstanding the action of the extraocular muscles and the internal force of the ciliary muscle.The latter mediates the change in lens shape in order to adjust the focusing power of the eye, and has been shown to have a very small and optically negligible effect on corneal shape. 1 A fundamental characteristic for corneal function is that of transparency.The cornea is classically accepted as a five-layered structure: an epithelial and an endothelial layer at the anterior and posterior surfaces respectively; two membranes: Bowman's under the epithelium and Descemet's anterior to the endothelium; and a central and predominant stroma composed of collagen fibrils, which represent approximately 90% of corneal thickness.
7][8][9] The regular arrangement of the fibrils within each layer is considered to be responsible for the transparency of the tissue. 2,10,11It is also an important factor for determining the mechanical properties of the cornea 12,13 as well as maintaining its shape. 14,15uch an organization contributes to characteristic patterns that are linked to highly ordered structures, such as crystals, which possess the property of birefringence.
These characteristic patterns seen when the structure is illuminated between crossed polarizers are a dark cross, the arms of which have been called isogyres, and colored rings or isochromatics.0][21][22][23][24] This cannot be explained on the same basis as crystals, given that ocular tissue is not highly ordered, but rather accords with the descriptions for elongated structures that arise in biology, such as cell layer arrangements. 25,26The lamellar organization of the corneal stroma is akin to a Wiener body, 27 which gives rise to form birefringence due to the directional variations in refractive index: i.e., differences along single fibers and across fiber layers. 27It has also been shown that isogyres can be formed in curved structures made of amorphous materials that do not possess any birefringent properties. 21,28The number of studies that have considered corneal birefringence show differences in findings, [19][20][21][22][23][24] and indeed it has been suggested that the random orientation of the central layers of the stroma results in no birefringence effects at the corneal apex. 8Such variation is to be expected in a biological tissue that differs in shape and thickness between individuals and changes with age and pathologies.There has been a paucity of investigation into the use of polarization optics for the study of stromal structure.Stromal orientation is important for optical quality and differences between individuals may suggest that the particular orientation of lamellae is optimized for image quality.In cases of corneal transplantation, the orientation of the donor cornea should therefore be considered.This study presents results of measurements on in-vivo corneal tissue using a new device that can determine the polarization properties of the cornea in vitro or in vivo.The instrument applies specialized software to determine the orientation of the corneal fibrils, offering the prospect of a detailed structural analysis which may have applications for clinical studies.

Scope
Development of a new non-invasive method to objectively evaluate the optical properties of the stroma, based on the interferometric analysis of diffractive and polarizing effects related to the birefringent properties of the stroma.

Method
Comparison between polarimetric interferometry image obtained by the interference between polarized light and stromal structure of human corneas using a new patented 29 medical device called Lumaxis® (Phronema SRL; Bari, Italy) with data published in the literature and obtained by x-ray, and second and third harmonic generation technique (SGH eTGH ).

Results
Data obtained by the polarimetric interferometry showed a cross-like pattern (isogyre), shown in Figure 1, which perfectly correlates with the pattern obtained by the x-ray, THG, and SHG analysis (Figs. 2 and 3), confirming that deep stroma lamellae have two preferential alignments along the superior-inferior and nasal-temporal directions.The regular distribution of stromal lamellae allows the stroma to behave as a polarizer, which eliminates the diffractive effect of the light during its journey into the stroma.

Conclusions
In accordance with the importance of regularity and orientation of stromal lamellae distribution in the corneal refractive process, the importance of polarimetric interferometry as a non-invasive technique to detect such orientation as a consequence of correlation/decorrelation between probe light polarization plane angle and stromal lamellae orientation becomes evident.Information from the cross-like pattern can be used for multiple applications in ophthalmology, such as corneal transplantation and diagnosis of corneal diseases due to stromal pathologies.This new technique represents a unique method to correlate the internal stromal structure and optical properties of the cornea.

Fig. 2 .
Fig. 2. (a) Contour maps of aligned collagen x-ray scatter from a left/right pair of normal human corneas .Note the high degree of body-line mirror symmetry.(b) Theoretical model of fibrillar arrangement based on (a).Reproduced from Boote et al.30

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Fig. 2. (a) Contour maps of aligned collagen x-ray scatter from a left/right pair of normal human corneas .Note the high degree of body-line mirror symmetry.(b) Theoretical model of fibrillar arrangement based on (a).Reproduced from Boote et al.30

Fig. 3 .
Fig. 3. (a) Contour map of aligned collagen x-ray scatter (a.u.) from a right human cornea.Superior, s, and nasal, n, positions are marked.Broken line denotes the limbus.Note the skewed diamond shape of the scatter contours, which displays mirror symmetry between the left and right eyes.(b) Proposed model of collagen fibril arrangement to explain the shape of the aligned scatter contours.The peripheral, oblique cornea is reinforced by chords of anchoring collagen of scleral origin.Figure modified from Boote et al.31