Non-invasive and label-free follow-up of accelerated-crosslinking using multiphoton tomography

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.