Three‐dimensional mapping of the orientation of collagen corneal lamellae in healthy and keratoconic human corneas using SHG microscopy

Abstract

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.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 secondharmonic 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 microscopy for the early diagnosis of keratoconus.SHG image acquired with sagittal optical sectioning (A) of a healthy cornea and (B) of a keratoconic cornea. Scale bars: 30 μm.

Self‐assembling amyloid‐like peptides as exogenous second harmonic probes for bioimaging applications

“…In particular, collagen I and collagen II have been proven to efficiently produce SHG signals . SHG has been applied to investigate rat and human corneal samples . Collagen fibrils in cornea were observed under SHG microscopy without any labelling.…”

Collagen fiber changes related to keratoconus with secondary corneal amyloidosis

“…Additionally, direct evidence for collagen fiber change has been the differing distribution of collagen orientation as observed by the synchrotron X-ray scattering pattern4 and the quantitative analysis of collagen lamellae in keratoconus, as observed by harmonic generation imaging microscopy 5. Marcatelli et al17 reported that the orientation of sutural lamellae close to Bowman’s membrane is significantly different between healthy and keratoconic samples. This suggests that the collagen structure in the keratoconic cornea differs greatly from that in the normal cornea.…”

Nonlinear optical imaging techniques (NLO) for painting investigation

“…NLO measurements were performed using a custom-made laser-scanning nonlinear microscope, originally developed by Cicchi R. et al for biological applications at INO-CNR [10]. The excitation source is a mode-locked Ti:Sapphire laser (Mira 900 F, Coherent Inc., Santa Clara, CA, US).…”

“…In Two-Photon-Excited Fluorescence (TPEF) the detection of fluorescence emitted after the simultaneous absorption of two infrared photons [9] yields to the identification of chromophores. Finally, fluorescence lifetime imaging microscopy (FLIM) measures time-decay of the fluorescence intensity exhibited by fluorescent emitting molecules [10], yielding information on the molecular microenvironment of a molecule and on its energy exchanges.…”

“…In this work we present the preliminary results obtained through the application of NLO imaging techniques with a custom-made laser-scanning non-linear micro-scope developed by INO-CNR (Cicchi R. et al) [10]. The analyses were performed on a series of single-and multi-layers systems, specifically designed for NLO imaging applications, simulating the real egg-tempera wood panel paintings.…”