In clinical practice, ectatic disorders, such as keratoconus, are treated by accelerated corneal collagen crosslinking (ACXL). The treatment is based on the photodynamic reaction of riboflavin with ultraviolet A (UVA) light and increases the cornea’s mechanical stability. The clinical outcome of ACXL is usually evaluated several weeks post-treatment. An earlier evaluation could lead to a faster re-intervention in case of failure which could avoid additional discomfort and pain for the patient. We propose multiphoton tomography (MPT) to evaluate the outcome of ACXL soon after treatment.
In this study, we investigate ACXL-induced changes to the cornea autofluorescence (AF) using MPT. ACXL was performed in de-epithelialized corneal donor buttons and keratoconus corneas by infusing the samples with 0.1% riboflavin solution followed by UVA irradiation using either an in-house adapted system or a commercial ACXL system. AF lifetime images of the tissue were acquired prior and after treatment using MPT. As a control, corneas without treatment were monitored at the same time points.
Higher AF lifetimes were observed in the stroma of treated corneas than in control samples. The stroma AF lifetime was higher anteriorly, corresponding to the area where ACXL was most effective. First changes were observed as soon as 2 ℎ after treatment. We demonstrate that MPT can be used to follow-up the outcome of ACXL and that ACXL-induced changes can be detected sooner than with conventional methods and non-invasively.
The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cells’ metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cells’ morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cells’ metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases.
Multiphoton tomography (MPT) is a novel label-free clinical imaging method for non-invasive tissue imaging with high spatial (300 nm) and temporal (100 ps) resolutions. In vivo optical histology can be realized due to the nonlinear excitation of endogenous fluorophores and second-harmonic generation (SHG) of collagen. Furthermore, optical metabolic imaging (OMI) is performed by two-photon autofluorescence lifetime imaging (FLIM). So far, applications of the multiphoton tomographs DermaInspect and MPTflex were limited to dermatology. Novel applications include intraoperative brain tumor imaging as well as cornea imaging. In this work we describe two-photon imaging of ex vivo human corneas unsuitable for transplantation. Furthermore, the cross-linking (CXL) process of corneal collagen based on UVA exposure and 0.1 % riboflavin was studied. The pharmacokinetics of the photosensitizer could be detected with high spatial resolution. Interestingly, an increase in the stromal autofluorescence intensity and modifications of the autofluorescence lifetimes were observed in the human corneal samples within a few days following CXL.
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