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.

Fig. 1

(a) Blue-IRIS mounting scheme for intact rabbit globe. Globes were mounted on a three-axis translation system during raster scanning. (b) Custom-built MZI used for phase change measurement. The reference arm and test arm were recombined to form an interference pattern that contained the phase difference between two arms. (c) Two-photon microscopy system used for TPEF and SHG imaging. Immediately after each TPEF and F-SHG image stack was collected, the filter (525 nm, 100 nm bandwidth) for the backward channel was replaced with a 405 nm, 30 nm bandwidth emission filter and another SHG image stack was taken from exactly the same ROI.

Fig. 2

(a) Histogram of one IRIS pattern written at 40 mm/s, its TPEF image is shown at the top right corner. (b) Curve fitting of the histogram.

Fig. 3

Interferogram and retrieved phase map of eye 3 taken from the MZI, with scan speeds of each pattern given on the interferogram.

Fig. 4

Phase change measured at 633 nm as a function of scan speed, repeated on three eyes (red, blue and green data points), with a linear fit and R² values included in the graph. The green “damage” line indicates that at speeds below 20 mm/s, which include 10 mm/s, damage is obtained, rather than a measurable phase change.

Fig. 5

TPEF (green), F-SHG (red) and B-SHG (magenta) images of the same ROIs over different IRIS patterns written at different speeds from eye 1. Note the faint green autofluorescence of the corneal epithelium on the top of all TPEF images, which contrasts with the strong TPEF signal emanating from the IRIS patterns. Note also the decreased SHG signal coincident with each IRIS pattern. Scale bar: 100 µm for all images.

Fig. 6

Change in TPEF (a), F-SHG (b), B-SHG intensity (c), and F/B ratio (d) as a function of IRIS scan speed. Linear least-square fitting was performed using all the data points from three eyes, with R² values indicated in each graph.