Tissue effects of intra-tissue refractive index shaping (IRIS): insights from two-photon autofluorescence and second harmonic generation microscopy

Abstract

Intra-tissue refractive index shaping (IRIS) is a novel, non-ablative form of vision correction by which femtosecond laser pulses are tightly focused into ocular tissues to induce localized refractive index (RI) change via nonlinear absorption. Here, we examined the effects of Blue-IRIS on corneal microstructure to gain insights into underlying mechanisms. Three-layer grating patterns were inscribed with IRIS ~180 µm below the epithelial surface of ex vivo rabbit globes using a 400 nm femtosecond laser. Keeping laser power constant at 82 mW in the focal volume, multiple patterns were written at different scan speeds. The largest RI change induced in this study was + 0.011 at 20 mm/s. After measuring the phase change profile of each inscribed pattern, two-photon excited autofluorescence (TPEF) and second harmonic generation (SHG) microscopy were used to quantify changes in stromal structure. While TPEF increased significantly with induced RI change, there was a noticeable suppression of SHG signal in IRIS treated regions. We posit that enhancement of TPEF was due to the formation of new fluorophores, while decreases in SHG were most likely due to degradation of collagen triple helices. All in all, the changes observed suggest that IRIS works by inducing a localized, photochemical change in collagen structure.

Nonlinear optical crosslinking (NLO CXL) for correcting refractive errors

“…(Wozniak et al, 2017;Xu et al, 2011) While the underlying principle of this technique remains poorly understood, denaturation of the fibrillar collagen seems likely given the finding that the non-centrosymmetric structure of collagen is lost following Blue-IRIS, as evidenced by the loss of second harmonic generated signals from fibrillar collagen. (Yu et al, 2019) How this affects the long-term stability of the change in refractive index, and what other components of the extracellular matrix are thermally denatured remains to be determined.…”