Noninvasive intratissue refractive index shaping (IRIS) of the cornea with blue femtosecond laser light

Abstract

Purpose: To test the feasibility of intratissue refractive index shaping (IRIS) in living corneas by using 400-nm femtosecond (fs) laser pulses (blue-IRIS). To test the hypothesis that the intrinsic two-photon absorption of the cornea allows blue-IRIS to be performed with greater efficacy than when using 800-nm femtosecond laser pulses.

Methods: Fresh cat corneas were obtained postmortem and cut into six wedges. Blue laser pulses at 400 nm, with 100-fs pulse duration at 80 MHz were used to micromachine phase gratings into each corneal wedge at scanning speeds from 1 to 15 mm/s. Grating lines were 1 μm wide, 5 μm apart, and 150 μm below the anterior corneal surface. Refractive index (RI) changes in micromachined regions were measured immediately by recording the diffraction efficiency of inscribed gratings. Six hours later, the corneas were processed for histology, and TUNEL staining was performed to assess whether blue-IRIS causes cell death.

Results: Scanning at 1 and 2 mm/s caused overt corneal damage in the form of bubbles and burns. At faster scanning speeds (5, 10, and 15 mm/s), phase gratings were created in the corneal stroma, which were shown to be pure RI changes ranging from 0.037 to 0.021 in magnitude. The magnitude of RI change was inversely related to scanning speed. TUNEL staining showed cell death only around bubbles and burns.

Conclusions: Blue-IRIS can be performed safely and effectively in living cornea. Compared with near-infrared laser pulses, blue-IRIS enhances both achievable RI change and scanning speed without the need to dope the tissue with two-photon sensitizers, increasing the clinical applicability of this technique.

Figures

Figure 1.

Blue-IRIS experimental setup. (A) Blue femtosecond laser pulses were focused through a 1.0-NA water-immersion objective, into living corneal tissue sandwiched between a glass slide and a no. 1 glass coverslip, and bathed in storage medium (Optisol-GS; Bausch & Lomb). The whole slide assembly was then mounted on a 3D scanning platform. (B) Blue-IRIS gratings inside a corneal wedge. Five sets of grating lines were micromachined into most pieces. The two outermost lines of each grating line set were micromachined at 0.5 mm/s to create reference damage marks, and the central lines were drawn at the indicated speeds.

Figure 2.

Phase-contrast micrographs of blue-IRIS grating lines in excised cornea. The central lines of each grating set were micromachined at scanning speeds of 1, 2, 5, 10, and 15 mm/s. The two outermost lines of each grating set (arrows) were micromachined at 0.5 mm/s to cause damage. For scanning speeds above 5 mm/s (C, D, E), clear, undamaged IRIS lines predominated within the line set, whereas lower scanning speeds (A, B) caused plasma luminescence and blebs within the line set.

Figure 3.

Using diffraction patterns to measure RI change attained with blue-IRIS. (A) Diffraction image of a blue-IRIS grating micromachined at 5 mm/s in a piece of live cornea. (B) Plot of first-order diffraction efficiency versus corresponding RI change. For the grating written at 5 mm/s, first-order diffraction efficiency was measured relative to the 0th order, The corresponding RI change is plotted in the figure with horizontal error bar (light gray).

Figure 4.

TUNEL staining after the blue-IRIS procedure. (A) Micrograph of a TUNEL-stained section of a corneal piece schematically shown in Figure 1B, with the forceps crush site at the corneal rim. Red: TUNEL-positive staining; blue: nuclear DAPI counterstain. Pink cells are both TUNEL- and DAPI-positive. Note the tissue disruption induced by the crush and the large number of TUNEL-positive cells (arrows) in the epithelium and stroma. Stromal cells mostly possessed the morphology of keratocytes. (B) Adjacent region of the same corneal slice, showing strong, diffuse, TUNEL-positive staining of IRIS line set A, created at 1 mm/s. This line set caused bubbles of damage in the micromachined region (see Fig. 2A). Note, however, the lack of TUNEL-positive staining in areas of stroma and epithelium directly above and below the micromachined (damaged) line set. Cross section of line set B, created at 2 mm/s with flanking (damage) lines at 0.5 mm/s shows TUNEL-positive cells only in regions of stroma crossed by the flanking burn lines, not the intervening IRIS lines. (C) Distal region of the same corneal slice shown in (A) and (B), illustrating TUNEL staining for line sets (C–E), inscribed at 5, 10, and 15 mm/s, with flanking (damage) lines written at 0.5 mm/s. Once again, a general pattern is observed, whereby TUNEL-positive cells are observed wherever flanking (damage) lines intersect stromal keratocytes. Only one TUNEL-positive cell is seen in the middle of a pure RI change line set (set C, 5 mm/s). All other line sets are free of such cells, suggesting that blue-IRIS, when it induces RI changes in a living cornea, generally does not cause a significant amount of cell death.