1. Introduction

Femtosecond laser technology has provided a powerful tool for corneal refractive surgery due to its minimally invasive nature and highly localized interaction with ocular tissue. Unlike traditional excimer lasers used in corneal surgery, femtosecond lasers can penetrate through corneal tissue and only affect targeted regions, without collateral damage, when focused tightly in the tissue. In 1999, a near-infrared (NIR) femtosecond laser with µJ pulse energy was first used for corneal flap cutting, keratomileusis, and intrastromal vision correction, offering advantages over conventional excimer lasers [1]. Later, NIR femtosecond laser pulses with nJ pulse energy were used for intra-tissue surgery with sub-micron precision [2]. These previous results were all based on destructive effects of the laser-tissue interaction, which were applied to corneal flap cutting [3–6]. In 2008, we first showed that a NIR femtosecond laser could be used for Intra-tissue Refractive Index Shaping (IRIS) inside lightly-fixed corneal tissue [7]. The initial IRIS was done using a low-pulse-energy femtosecond laser at 800 nm. This NIR femtosecond IRIS process induced refractive index (RI) changes averaging 0.008, but this required an extremely slow scanning speed of only 0.7 μm/s [7]. The relatively small refractive changes attained and extremely low speeds in this initial work made it impractical for clinical applications. In this report, we show how the effectiveness of IRIS can be significantly increased with both exogenous and endogenous two-photon absorption enhancement.

2. Experimental setup

The experimental setup for Intra-tissue Refractive Index Shaping (IRIS) is shown in Fig. 1 . A mode-locked Ti:Sapphire laser (Mai Tai, Spectra Physics), which generates pulses of 100 fs pulse width, with an 80 MHz repetition rate, and a tunable spectrum of 780~920 nm was used. The average output power of the laser at 800 nm was ~0.8 W, but was attenuated to a proper level (~80mW) by passing through a variable metallic attenuator. We compared two different modes of IRIS: Blue-IRIS and NIR-IRIS. The first mode of the system (Blue-IRIS) works when both flip mirrors are out of the beams. With some beam shaping, the NIR laser pulses at 800 nm were delivered to a BBO crystal for second harmonic generation (SHG), converted to blue femtosecond pulses at 400 nm, and finally focused through a high numerical aperture (NA) water immersion objective (20X, NA 1.0, W Plan-Apochromat, Carl Zeiss MicroImaging GmbH) into the corneal tissue at a depth of ~150 μm. The corneal tissue was sandwiched between a glass slide and a #1 coverslip, and mounted on a three-dimensional, linear motor stage system, consisting of three DC motor stages (VP-25XA, Newport Corporation). The second mode of the system (NIR-IRIS) works when the flip mirrors are in the beams, and NIR laser pulses are delivered directly to the sample. The entire laser micromachining process was monitored via a CCD camera. After the femtosecond laser micromachining procedure, the corneal tissue was imaged using a phase contrast microscope (BX51, Olympus). A custom-built scatterometer was then used to capture the diffraction pattern of the grating written inside the corneal tissue, which enabled measurement of refractive index changes induced along the grating lines.

 

Fig. 1 Experimental setup for Intra-tissue Refractive Index Shaping (IRIS). When the flip mirrors are in, the near-infrared (NIR) laser pulses at 800 nm passes through the system, and the procedure is called NIR-IRIS. When the flip mirrors are out, NIR laser pulses are converted to blue laser pulses at 400 nm, and the procedure is termed Blue-IRIS.

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3. Experimental procedure, results and discussion

The samples used in this study consisted of feline corneas (Liberty Research Inc.), stored in Optisol-GS (Bausch&Lomb, Inc) at 4°C overnight following extraction. Optisol-GS is a special corneal storage medium used in corneal transplant operations, which can keep extracted corneas alive for up to 14 days [8,9]. The cornea absorbs primarily in the ultraviolet and transmits most of the visible and near-infrared (NIR) light. When laser pulses are tightly focused in a small volume inside the corneal stroma, it is possible to induce multi-photon absorption due to the high laser intensity. Using an Ocean Optics spectrometer (HR4000), we measured the transmission spectrum of living corneal tissue either undoped or doped with sodium fluorescein (Na-Fl), commonly used in ophthalmic practice, by soaking the cornea into Na-Fl solution for ~2 hours. As shown in Fig. 2 , the native cornea has almost no direct one-photon absorption at the laser excitation wavelength (800 nm) and also very weak two-photon absorption (TPA), for the NIR-IRIS process. When Na-Fl is doped into the cornea, the TPA of the corneal tissue is enhanced. Figure 3A gives one example of NIR-IRIS performed in living corneal tissue doped with 1% Na-Fl. The RI change attained was ~0.012 at a scanning speed of 2 mm/s. A range of scanning speeds and Na-Fl doping concentrations were tested, and the results are plotted in Fig. 4 (NIR-IRIS with Na-Fl doping) [10]. Thus, NIR-IRIS can be significantly enhanced by doping the tissue with exogenous TPA sensitizers.

 

Fig. 2 Effect of Sodium Fluorescein (Na-Fl) doping (1%) on the transimission spectrum of living corneal tissue, stored in Optisol-GS solution. Note how Na-Fl doping decreases transmissivity of the cornea in the 300-500nm wavelength range, which corresponds well with the absorption spectrum of Na-Fl.

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Fig. 3 (A): NIR femtosecond IRIS (NIR-IRIS) in living corneal tissue doped with 1% Na-Fl. Scanning speed is 2 mm/s, and pulse energy is ~1.5 nJ. Refractive index change is ~0.012. Damage lines on two sides (arrow) are for identification purposes only. (B): Blue femtosecond IRIS (Blue-IRIS) in native live cornea. Scanning speed is 5 mm/s, and pulse energy is ~1 nJ. Refractive index change is ~0.037. Damage lines on two sides (arrow) are for identification purposes only.

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Fig. 4 Comparison of refractive index changes attainable in live cornea induced by Blue-IRIS and NIR-IRIS. Measures are provided as a function of scan speed. Blue-IRIS was performed in undoped corneal tissue only, while NIR-IRIS data was obtained in corneal tissues doped with various concentrations of Sodium Fluorescein (Na-Fl).

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However, while NIR-IRIS induced greater RI change at much faster scanning speeds in living cornea doped with Na-Fl, as compared to IRIS in fixed cornea, the process relied on exogenous doping with Na-Fl to increase efficacy. In living tissue, the corneal epithelium acts like a barrier and needs to be scraped off to allow exogenous Na-Fl to penetrate into the region of corneal stroma targeted for IRIS. Epithelial removal creates a surface wound and causes a wound healing response in the cornea, which decreases optical quality and creates significant complications for both live animal studies and human applications. This motivated our work on Blue-IRIS, which uses femtosecond laser pulses at 400 nm to induce RI changes in the cornea directly, without the need for epithelial removal or exogenous TPA enhancement. Blue-IRIS achieves even better optical results in native, living corneas (Fig. 3B) than NIR-IRIS with Na-Fl doping. The mean RI change attained at a scanning speed of 5 mm/s was 0.037 ± 0.0005. The smallest RI change attained was 0.021 ± 0.001 at a scanning speed up to 15 mm/s (Fig. 4). For comparison, the maximum RI change attained when performing NIR-IRIS with Na-Fl doping was 0.02 at a scanning speed of 0.5 mm/s. Thus, a similar RI change can be induced with Blue-IRIS at a scanning speed 30X faster than with NIR-IRIS. The maximum achievable RI change was also significantly increased.

Our hypothesis for the larger effects of Blue-IRIS is that there is stronger TPA in the native cornea when using blue femtosecond laser pulses at 400 nm than when using 800 nm pulses. To verify this hypothesis, we performed a direct measurement of the nonlinear absorption of native corneal tissue without any exogenous doping. The corneal tissue was kept in Optisol-GS solution during the entire measurement process to prevent dehydration and opacification which can significantly affect the absorption properties of cornea [11,12]. The blue femtosecond laser pulses at 400 nm were first focused outside the corneal tissue, and the transmitted power through the whole sample was measured. Then the laser focus was moved into a region ~150 μm below the corneal anterior surface, and the transmitted power was measured again. This procedure was repeated for various input powers, and the difference of the transmitted laser powers between the in- and out-of-focus cases reveals the endogenous nonlinear absorption in corneal tissue when there is no doping. Figure 5 shows the transmitted power when the laser is either in-focus or out-of-focus with respect to the corneal tissue. When the input power is low, there is very little nonlinear absorption in the native corneal tissue, however when the input power increases, we can observe more and more nonlinear absorption, which appears as a departure from linear transmitted power. To understand this better, we also plotted the nonlinear absorption as a function of input power (or intensity when the focusing condition remains the same, as shown in Fig. 6 ). The absorption alpha was obtained as $\alpha =Ln(P_{in}/P_t)/L$, where $P_{in}$ and $P_t$ are input and transmitted laser power, and $L$ is the confocal parameter, respectively. The nonlinear absorption appeared to be linear with respect to input laser intensity, which is true when TPA is the dominant form of nonlinear absorption. The TPA coefficient $\beta$ of native cornea was calculated as $\beta =\alpha /I$, and was estimated to be ~8 × 10⁻⁶ cm/W, when using a femtosecond laser at 400 nm [13]. Compared to the measurement of TPA in hydrogel polymers doped with fluorescein [14], the nonlinear absorption coefficient in native corneal tissue with blue femtosecond laser pulses was roughly twice as large as that in doped hydrogel polymers. Fluorescein is also used as exogenous doping for NIR-IRIS. It is then conceivable that the efficiency of Blue-IRIS with endogenous TPA was higher than that of NIR-IRIS with exogenously enhanced TPA, in terms of both achievable refractive index change and the corresponding scanning speeds.

 

Fig. 5 Transmitted versus input blue femtosecond laser power in native live corneal tissue (i.e. without any doping). Note the increased nonlinear absorption of the tissue (computed as increasing difference in transmitted power between in-focus and out-of-focus conditions) as input power is increased above ~30mW.

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Fig. 6 Nonlinear absorption in native live corneal tissue as a function of input laser power.

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4. Conclusion

To summarize, we studied and characterized two different means of enhancing Intra-tissue Refractive Index Shaping (IRIS) based on femtosecond laser micromachining. Both NIR-IRIS with exogenous TPA enhancement and Blue-IRIS with endogenous TPA enhancement demonstrate significant improvement over the original IRIS procedure in undoped, lightly fixed corneal tissue. Comparison of these three methods shows that exogenous doping of the cornea with Na-Fl increases the effectiveness of NIR-IRIS, but is less advantageous clinically due to the need for epithelial removal. Blue-IRIS utilizes the endogenous two-photon absorption of the cornea using femtosecond laser pulses at 400 nm, and creates large refractive index changes at high scanning speeds without the need for doping with exogenous substances or for disruption of the corneal epithelium. This makes Blue-IRIS the least invasive of all current refractive surgical procedures, such as LASIK and PRK.