December 2008
Volume 49, Issue 12
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Cornea  |   December 2008
Intratissue Refractive Index Shaping (IRIS) of the Cornea and Lens Using a Low-Pulse-Energy Femtosecond Laser Oscillator
Author Affiliations
  • Li Ding
    From The Institute of Optics, the
  • Wayne H. Knox
    From The Institute of Optics, the
  • Jens Bühren
    Department of Ophthalmology, and the
    Center for Visual Science, University of Rochester, Rochester, New York.
  • Lana J. Nagy
    Department of Ophthalmology, and the
    Center for Visual Science, University of Rochester, Rochester, New York.
  • Krystel R. Huxlin
    Department of Ophthalmology, and the
    Center for Visual Science, University of Rochester, Rochester, New York.
Investigative Ophthalmology & Visual Science December 2008, Vol.49, 5332-5339. doi:10.1167/iovs.08-1921
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      Li Ding, Wayne H. Knox, Jens Bühren, Lana J. Nagy, Krystel R. Huxlin; Intratissue Refractive Index Shaping (IRIS) of the Cornea and Lens Using a Low-Pulse-Energy Femtosecond Laser Oscillator. Invest. Ophthalmol. Vis. Sci. 2008;49(12):5332-5339. doi: 10.1167/iovs.08-1921.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To assess the optical effect of high-repetition-rate, low-energy femtosecond laser pulses on lightly fixed corneas and lenses.

methods. Eight corneas and eight lenses were extracted postmortem from normal, adult cats. They were lightly fixed and stored in a solution that minimized swelling and opacification. An 800-nm Ti:Sapphire femtosecond laser oscillator with a 27-fs pulse duration and 93-MHz repetition rate was used to inscribe gratings consisting of 20 to 40 lines, each 1-μm wide, 100-μm long, and 5-μm apart, 100 μm below the tissue surface. Refractive index changes in the micromachined regions were calculated immediately and after 1 month of storage by measuring the intensity distribution of diffracted light when the gratings were irradiated with a 632.8-nm He-Ne laser.

results. Periodic gratings were created in the stromal layer of the corneas and the cortex of the lenses by adjusting the laser pulse energy until visible plasma luminescence and bubbles were no longer generated. The gratings had low scattering loss and could only be visualized using phase microscopy. Refractive index changes measured 0.005 ± 0.001 to 0.01 ± 0.001 in corneal tissue and 0.015 ± 0.001 to 0.021 ± 0.001 in the lenses. The gratings and refractive index changes were preserved after storing the micromachined corneas and lenses for 1 month.

conclusions. These pilot experiments demonstrate a novel application of low-pulse-energy, MHz femtosecond lasers in modifying the refractive index of transparent ocular tissues without apparent tissue destruction. Although it remains to be verified in living tissues, the stability of this effect suggests that the observed modifications are due to long-term molecular and/or structural changes.

Conventional ultraviolet nanosecond excimer lasers have been successfully used for corneal refractive surgery, including photorefractive keratectomy (PRK), laser-assisted in situ keratomileusis (LASIK) and laser subepithelial keratomileusis (LASEK). By ablating corneal tissue through direct, 1-photon absorption of ultraviolet light, these lasers alter the curvature, thickness, and ultimately the optical power of the cornea. 1 2  
The rapid development of femtosecond laser technology has provided an additional tool for corneal refractive surgery. In contrast to the photoablative ultraviolet lasers, femtosecond laser pulses in the near infrared can pass through transparent corneal tissue without significant 1-photon absorption. Only when pulses are focused inside the cornea, is the intensity of the beam sufficient to cause nonlinear, typically multiphoton absorption, and a range of modifications to the tissue. Because the absorption is strongly nonlinear, the laser-affected region tends to be highly localized, leaving the surrounding region unaffected, or minimally affected. 3 4 5 This unique capability for three-dimensional, high-precision micromachining is the primary reason for the introduction of femtosecond lasers to refractive surgery, where their main application has been in corneal flap cutting. 6 7 8 9 10 11 12 For this application, femtosecond laser pulses with a low repetition rate (Hz–kHz range) are used to induce photodisruption and destructive, optical breakdown of corneal tissue. This is generally associated with high-density microplasma generation, bubble formation, and shock-wave emission, often extending beyond the focal region. Compared with mechanical blade microkeratomes, femtosecond lasers are better able to define the depth of the cut, eliminating some flap-related complications and generally improving visual outcomes. 13 14 15 16 However, like any cut, the creation of a femtosecond laser flap causes biomechanical changes in the cornea, and since tissue is destroyed, a wound-healing reaction ensues. 13 17 18 This wound-healing reaction includes regeneration of the protective corneal epithelium and the differentiation of the usually quiescent and supportive stromal keratocytes into reactive, inflammatory, and contractile myofibroblasts. 19 20 Myofibroblasts appear responsible for most of the negative side effects of laser refractive surgery, including haze or loss of corneal transparency, and unintended changes in corneal shape, which negatively impact the optical quality of the eye. 19 21 Although increasingly popular, femtosecond laser flap-cutting remains limited by its high cost, accessibility, and uncertainty about its long-term photochemical, mechanical, and biological effects. 17 22 Recent reports have detailed negative side effects of this technique, particularly in terms of tissue destruction, which, at some laser settings, appears stronger than after mechanical microkeratome cuts. 17 22  
The ability to alter corneal shape or optics without causing tissue destruction (and thus, a wound-healing response), would significantly improve laser refractive correction by decreasing or eliminating the negative side effects that currently compromise optical outcomes and ocular health. To date, most clinical femtosecond lasers use micro- or millijoule pulses with a low repetition rate (Hz–kHz range) and spot diameters in the range of several micrometers. 6 7 This contrasts with femtosecond laser parameters that have been established for other biomedical applications. 3 High-repetition-rate (>1 MHz) femtosecond laser oscillators with pulse energies on the order of nanojoules have successfully been used to micromachine artificial media and perform cellular nanosurgery. 23 24 25 26 However, in clear ocular tissues, such as the cornea, nanojoule femtosecond laser pulses with 170-fs pulse duration and 80-MHz repetition rate have only been reported to induce destructive optical breakdown. 27 For such lasers, the diffusion time of nonlinearly absorbed laser energy is longer than the time interval between laser pulses. As a result, absorbed energy accumulates locally, with destructive consequences. 28  
In the present study, we reduced femtosecond laser pulse energies below the optical breakdown threshold of lightly fixed cat corneas and lenses to examine the optical consequences of the resulting tissue modifications. In both silicone and nonsilicone-based hydrogels, this approach induces a significant change in refractive index without visible plasma luminescence or bubble formation. 29 Changing the refractive index of the cornea or lens without tissue destruction, a phenomenon we termed Intra-tissue Refractive Index Shaping (IRIS), could represent a major advance in the field of laser refractive correction. 
Materials and Methods
Extraction and Preparation of Cat Corneas and Lenses
Eight corneas and eight lenses were extracted under surgical anesthesia from five normal, adult domestic short-hair cats (Felis cattus). All animal procedures were conducted in accordance with the guidelines of the University of Rochester Committee on Animal Research, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the NIH Guide for the Care and Use of Laboratory Animals. Cat corneas and lenses are similar to human corneas and lenses in histologic structure, molecular composition, and optical properties. 30 However, in contrast with the problems associated with obtaining postmortem human eyes, using cat eyes allowed us to precisely control postmortem extraction time and tissue processing parameters. To avoid decomposition and opacification before femtosecond laser micromachining, we immediately drop fixed the extracted cat tissues for 10 minutes (corneas) or 1 hour (lenses) in a solution consisting of 1% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS; pH 7.4). Lenses were then cut into 500-μm-thick slices with a HM650V vibratome (Microm International, Walldorf, Germany), after which lens sections and whole corneas (also ∼500 μm thick) were immersed in a mixture of 30% ethylene glycol and 30% sucrose in 0.1 M PBS (pH 7.4) at 4°C. Storage in this solution minimized tissue swelling and loss of transparency. Small pieces of tissue (∼1 cm2) were then flattened onto a clear glass slide (1 × 3 in., 1 mm thick; Surgipath Medical Industries Inc., Richmond, IL). In the case of corneal pieces, this procedure was performed with the epithelium facing up and the endothelium facing down. A glass coverslip (No. 0211 zinc titania glass; Corning, Corning, NY) was placed on the top of each piece, stabilizing it for the duration of the experiment. The ethylene glycol/sucrose storage solution was used as mounting medium to minimize dehydration of the cornea and lens tissue samples since these effects are known to alter the refractive index and transparency of both tissues. 31 32 33  
Femtosecond Laser Micromachining
Femtosecond laser micromachining was conducted as previously described in hydrogels. 29 The laser source was a Kerr-lens mode-locked Ti:Sapphire laser (K-M Labs, Inc., Boulder, CO). The laser oscillator generates pulses averaging 300 mW, 27 fs in duration, with a 93-MHz repetition rate at 800-nm wavelength. A continuously variable, metallic, neutral density filter was inserted into the optical path and used to adjust the incident laser power onto each cat cornea and lens piece. Pulses were focused 100 μm below the tissue surface using a 60×, 0.70 NA microscope objective (LUCPlanFLN; Olympus, Lake Success, NY) with an adjustable working distance of 1.5 to 2.2 mm. Because the large amount of glass within the microscope objective induced significant chromatic dispersion into the femtosecond laser pulses, broadening the pulse durations, a standard extracavity prism, double-pass configuration was used to compensate for the dispersion and maintain the ultrashort pulse duration. By carefully adjusting this dispersion compensator, we obtained near transform-limited 27-fs duration pulses at the focal point of the focusing objective, as measured by a collinear autocorrelator using third-order surface harmonic generation. 34 35 During IRIS, the slide containing the biological tissue samples was mounted on a 3-D scanning platform consisting of a scanning stage (P-622.2CD XY; Physik Instrumente, Karlsruhe/Palmbach, Germany) with 250 μm travel range and 0.7-nm close-loop resolution, and a linear servo z-axis scanning stage (VP-25XA; Newport, Mountain View, CA) with 25-mm travel range and 100-nm resolution. An infrared CCD camera was used to monitor the micromachining process and the generation of visible plasma luminescence in real time. 
Experiments were conducted at room temperature (∼25°C). It took approximately 40 minutes to create a 100 × 50-μm grating and conduct the immediate postmicromachining measurements. Corneal trimming and mounting did not exceed 10 minutes in duration, and the corneal tissue was exposed to ambient air during the trimming process for at most 2 minutes. Since we saw no significant changes in corneal and lens transparency or thickness at the end of our micromachining experiments, we concluded that our manipulations did not cause significant corneal or lenticular dehydration or swelling. 
The first experimental step was to establish thresholds for the optical breakdown of lightly fixed cat cornea and lens. The neutral-density filter was first adjusted to minimize the focused incident laser power on the cornea and the lens below their breakdown thresholds. 4 5 Adjusting the neutral density filter then progressively increased the incident laser power. The breakdown threshold power was reached when visible plasma luminescence suddenly appeared and strong light-scattering as well as laser-induced damage became visible (Figs. 1 2) . With our 0.70-NA long-working-distance objective, the measured breakdown thresholds for cat cornea and lens were ∼55- and 75-mW average laser power, respectively, which corresponds to pulse energies of 0.6 and 0.8 nJ. 
Once the tissue-breakdown thresholds were established, the focused laser power was lowered gradually by carefully adjusting the neutral-density filter until lines could be micromachined without the induction of bubbles or burns. Average laser power settings at which this could be done were 30 mW in the cornea and 45 mW in the lens, corresponding to pulse energies of approximately 0.3 and 0.5 nJ, respectively. These values lay between those used for imaging and our measured breakdown thresholds. The gratings were micromachined in the horizontal plane within the stromal layer of each corneal piece and the cortex of each lens at a constant speed of 0.7 μm/s for the cornea and 1 μm/s for the lens. The spherical aberration at the laser focus induced by refractive index mismatch was compensated by adjusting the correction collar of the focusing microscope objective to achieve the smallest possible laser-affected region along the laser propagation direction. 29  
Measurement of Refractive Index (RI) Change
To assess whether the gratings generated in corneal and lens pieces were associated with a change in RI, the slides containing the tissue were first placed under an Olympus BX51 optical microscope where gratings were localized under differential interference contrast (DIC) imaging. A low-power 632.8 nm He-Ne laser was then used to irradiate the gratings, generating a diffraction pattern that was captured by a digital camera and used to calculate RI changes attained as described previously. 29 In brief, a power meter measured the intensity of the zero- to third-order diffracted light from the gratings, and the different order diffraction efficiencies were obtained by calculating the ratios between the intensity of the first-, second-, and third-order and the zero-order diffraction light. Because the intensity distribution of the diffraction pattern of a phase grating is proportional to the square of the Fourier transform of the transmittance function of the grating, 36 one particular value of RI change matches only one particular diffraction efficiency value. 29 To reduce measurement error of the diffraction order intensities, we collected five measurements on each grating, calculating the average value obtained and its SD. In principle, the spatial distribution of the RI change within the micromachined region was a small-scale gradient-index structure. However, for the purpose of the present investigation, we presumed the index profile to be uniform within the grating lines, which were only 3 μm deep, because the spherical aberration at the focal point was corrected. 29  
The micromachined corneal and lens pieces were then stored in the ethylene glycol/sucrose solution at 4°C. After 1 month, each piece was remounted onto a new glass slide for imaging and a repeat of the diffraction light intensity measurements. This method allowed us to assess whether the RI change initially observed had been maintained during storage. 
Results
Patterns Created by Femtosecond Laser Pulses below the Optical Breakdown Thresholds
Exposure of lightly fixed cat corneal and lenticular tissue to 0.3- or 0.5-nJ femtosecond laser pulses (30 or 45 mW average laser power), respectively, resulted in the reliable creation of line gratings approximately 100 μm below the epithelial surface or 100 μm below the lens surface in all test samples (Figs. 3 4) . When imaged immediately after micromachining, individual grating lines could be clearly observed and distinguished with DIC microscopy, but they were practically invisible when viewed under bright-field transmission microscopy. This result could be interpreted as the grating lines’ having very low scattering properties, which is in contrast to the destructive tissue changes observed when laser energy was increased above the optical breakdown threshold (Figs. 1 2) . Using the knife-edge method, 37 we ascertained that the laser focus diameter was 2.5 μm in air, which was much bigger than the micromachined line widths. Thus, it appears that only the central part of the laser focal area had sufficient intensity to modify corneal and lens tissue. 
Effect of Low-Pulse-Energy Femtosecond Laser Micromachining on the RI of Cat Corneal Stroma and Lens
Because displacement of the stromal collagen lamellae as a result of postmortem corneal swelling could not be completely avoided, the scattering effect from the zero-order diffraction light was very strong obscuring the first-order diffraction light. 33 Thus, only the second- and third-order diffraction efficiencies of each grating could be measured and used to calculate an approximate RI change in corneal pieces (Fig. 5B) . Because tissue swelling and opacification were minimal in slices of lens cortex, the zero- through third-order diffraction light could be measured clearly (Fig. 6A) , and the first- and second-order diffraction efficiencies were used to calculate the induced RI change (Fig. 6B) . Although single-diffraction efficiency is usually sufficient to calculate the refractive index, we measured first/second or second/third combinations to confirm that the RIs calculated were consistent through different diffraction orders, assuming that the RIs of cat corneal stroma and lens cortex were 1.376 and 1.400, respectively. 30 For corneal stroma, the RI changes induced by the laser in our multiple samples ranged between 0.005 ± 0.001 and 0.01 ± 0.001 (Fig. 5B) . For cat lens cortex, RI changes (and scanning speeds) were larger, ranging between 0.015 ± 0.001 and 0.021 ± 0.001 (Fig. 6B)
Long-Term Maintenance of IRIS in Fixed Corneas and Lenses
After micromachining, each cornea and lens piece was stored in an aqueous solution for 1 month to determine whether IRIS could be maintained over the long term. After 1 month, the tissue pieces were removed from storage and reexamined. Our first observation was that although the storage solution significantly slowed corneal swelling and opacification, it did not completely prevent either. Despite this, DIC microscopy was able to reveal the grating structures initially micromachined (Figs. 7A 8A)
For both corneas and lens slices, the diffraction light distribution of 1-month-old gratings (Figs. 7B 8B)was no different from that obtained right after the gratings’ creation (Figs. 5B 6B) . In the corneal pieces, the scattering light from the zero order diffraction still obscured the first order diffraction. However, the second-, third-, and even fourth-order diffractions were visible and measurable. In the lens pieces, the first-, second-, and third-order diffractions were visible. The RI change after 1 month of storage ranged between 0.005 ± 0.001 and 0.01 ± 0.001 for corneal pieces and between 0.015 ± 0.001 and 0.021 ± 0.001 for lens slices. 
Discussion
The present study reports on the optical consequences of focusing a high-repetition-rate, low-pulse-energy femtosecond laser onto clear, biological tissues. Gratings were micromachined onto the stroma and cortex of excised, lightly fixed cat corneas and lenses. The inscription of gratings into these tissues induced small, but significant and persistent RI changes with low scattering loss, a phenomenon we termed IRIS. The main difference between the present results and previous uses of femtosecond lasers in corneal flap cutting is that our application was designed to avoid tissue destruction. We believe that this may be the first report in which the RI of the corneal stroma and lens cortex has been modified noninvasively with a femtosecond laser. 
Tissue-Breakdown Threshold Versus Pulse Duration and Repetition Rate
Choosing the right laser parameters was critical to performing IRIS in the cat cornea and lens. Not only did the femtosecond laser fluence at the objective focus have to be below the optical breakdown threshold of the tissue, it also had to be strong enough to induce some nonlinear changes. To achieve this, the average power of the incident laser beam was adjusted to lie between the power for nonlinear imaging (5–10 mW for 100-fs pulses, which causes no tissue disruption 3 38 ) and the breakdown threshold (80 mW for corneas when using 170-fs pulses and 1.30-NA focusing 27 ; 55 mW for corneas and 75 mW for lenses when using 27-fs pulses and 0.70-NA focusing in the present study). 
In the past two decades, extensive experimental and theoretical work has been done to characterize laser-induced optical breakdown thresholds in different materials, including corneal tissue 3 4 5 39 40 41 42 43 and lens. 44 45 46 47 With the exception of the study by Krueger et al., 47 most of this work centered on continuous-wave (CW) lasers or on single pulses from low-repetition-rate lasers in which thermal diffusion time is much shorter than the time interval between adjacent pulses. Thus, each pulse is responsible for a change in the tissue. Indeed, it has been established that for pulses longer than 10 ps, the optical breakdown threshold fluence scales as the square root of the pulse duration. 42 For pulses shorter than 10 ps but longer than approximately 100 fs, the experimental results show a departure from this dependence. However, whether threshold fluence increases or decreases as pulse durations get shorter remains a challenging question. 39 41 43 Some models predict that the threshold would first increase, then decrease when pulse duration becomes shorter than 100 fs, but there is no solid experimental evidence to support this. 43 More recently, it has been claimed that for corneal stroma, the breakdown threshold is almost plateaulike when the pulse duration is between 100 fs and 1 ps, with a rapid decrease in threshold for pulse durations in the low end of the femtosecond range. 5 However, insufficient experimentation on cornea and lens using sub-100-fs pulses makes it difficult to support this prediction; furthermore, existing data were collected using single pulses from low-repetition-rate lasers. 
When high-repetition-rate femtosecond laser pulses are used, cumulative, free-electron–mediated chemical effects, photochemical bond breaking, and thermal effects contribute to the laser-tissue interaction. As a result, the breakdown threshold fluence may be quite different from that predicted by current models. 3 Several studies on the effects of high-repetition-rate femtosecond lasers on fused silica and borosilicate glass have found that laser pulses greatly increased the temperature of the materials at the laser focus. 48 Vogel calculated the temperature change in water would be >10°K with a 0.6-NA focusing lens and 100-fs laser pulses, 3 assuming that with each pulse, an energy density of 1 J/cm3 at the center of the initial temperature distribution is deposited. In the present experiments, using very high-repetition-rate (93 MHz), ultrashort laser pulses (27 fs), we found the optical breakdown threshold for the 0.70-NA focusing condition in lightly fixed corneal stroma and lens cortex to be 55- and 75-mW average laser power, respectively. This corresponds to 0.6- and 0.8-nJ pulse energy, respectively, both lower than the optical breakdown power reported by König et al., 27 using 1-nJ pulse energy, 170-fs pulse duration, and 1.30-NA focusing in porcine corneas. By using 30- and 45-mW average laser power (0.3- and 0.5-nJ pulses), we were able to induce IRIS, without accompanying photodisruption and tissue destruction. 
Mechanisms of IRIS in the Cornea and Lens
Although the present results show that we can reproducibly attain small RI changes in lightly fixed corneas and lenses, they reveal little about the mechanism(s) likely to underlie these changes, other than that they depend critically on incident laser pulse energy, laser repetition rate and pulse duration. Although our laser pulses did not cause overt tissue damage, the possibility remains that they induced changes in extracellular, subcellular and molecular structure. König et al. 27 have in fact used femtosecond lasers to perform subcellular manipulations (transfections, intracellular nanosurgery) on live cells. 25 26 However, they did not discuss whether these manipulations were associated with optical changes, such as those reported in the present study. An important next step for our work is to test the feasibility of IRIS in living cornea and lens, where the mechanisms and magnitude of the effects may yet be different from those observed presently. 
Since 93-MHz femtosecond pulse trains and submicrometer/second scanning speeds were used in our experiments, each region of the gratings was exposed to millions of femtosecond laser pulses. The interval time between these successive pulses was approximately 11 ns, which is much shorter than the thermal diffusion time out of the focal volume. Thus, a cumulative thermal effect should be considered a possible mechanism of IRIS in the biological tissues tested. However, our data suggest that the cumulative thermal effect is likely localized within the focal volume, and has negligible impact on surrounding regions. As such, and compared to the overall thickness of the tissue (∼500 μm), micromachining is also unlikely to affect tissue thickness. 
Given the critical role played by hydration in maintaining the RI and clarity of the cornea and lens, 32 33 one possibility is that the femtosecond laser pulses used in the present study exerted their main optical effect by displacing water from the corneal or lenticular tissue. However, this is unlikely, because RI changes attained after micromachining were maintained even after storing the tissue pieces in an aqueous solution for 1 month. The stored corneas and lens sections swelled clearly over that period, suggesting that they were exchanging (and especially incorporating) water from the aqueous solution in which they were immersed. Thus, micromachining likely caused sustained molecular and/or structural changes in the collagen/proteoglycan extracellular matrix and/or the cells contained within the micromachined portions of tissue. Ongoing experiments with Raman spectroscopy, 49 and electron microscopy are attempting to ascertain the nature of these changes. 
Conclusions
The present study demonstrates, for the first time, that it is possible to cause low-scattering-loss RI modifications in lightly fixed cat cornea and lens using 93-MHz repetition rate, 27-fs laser pulses with 0.3- and 0.5-nJ pulse energies. These modifications are visible only using DIC microscopy and are not associated with apparent tissue damage. They represent RI changes between 0.005 ± 0.001 to 0.01 ± 0.001 for the corneal stroma and 0.015 ± 0.001 to 0.021 ± 0.001 for the lens cortex. Preservation of IRIS over a month of refrigerated storage suggests that the femtosecond laser-induced modifications are likely to involve relatively long-term molecular/structural alterations. 
Although the RI changes themselves were small, their impact on optical power was significant. Based on published values for the power (39 D) and native RI (1.376) of the cat cornea, 30 IRIS should generate a change in corneal power ranging between 0.14 and 0.28 D (assuming that the RI change affects the thickness of the cornea uniformly). Similarly, in the cat lens (power, 53 D; RI of the homogeneous lens, 1.554), 30 the RI changes induced by micromachining should theoretically alter lenticular power by between 0.5 and 0.7 D. However, the scanning speeds used (0.7–1 μm/s) made the micromachining process too slow for most practical applications. These observations and the fact that fixation and storage of ocular tissues postmortem are likely to affect the mechanisms and magnitude of RI changes attained, make it necessary to measure the impact of IRIS in living corneas and lenses. Our goal is not only to detail the cellular and molecular mechanisms underlying IRIS, but to further manipulate the size, placement and design of micromachined patterns, as well as the magnitude of the RI changes with which they are associated. The ability to alter the native RI of the cornea and lens without causing significant damage has important practical implications. Not only could this change our approach to laser refractive surgery, but also to vision correction in general. For instance, the preservation of tissue clarity during the treatment allows the application of IRIS for refractive corrections in a closed-loop approach (e.g., for the correction of higher-order aberrations). In conclusion, the feasibility of IRIS in clear ocular media demonstrated in the present study offers new possibilities for long-term, noninvasive alterations, marking or pattern inscription within biological tissues. 
 
Figure 1.
 
Femtosecond IRIS in lightly fixed cat corneal stroma just around the tissue-breakdown threshold. (A, C) DIC images of lines created in the stroma of two lightly fixed cat corneas with 0.6-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and “bubbles” along the micromachined lines (clear, horizontal lines within stromal tissue). (B, D) BF images of the corneal region in (A, C) illustrating the visibility of spots of tissue destruction (arrow) and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A, C).
Figure 1.
 
Femtosecond IRIS in lightly fixed cat corneal stroma just around the tissue-breakdown threshold. (A, C) DIC images of lines created in the stroma of two lightly fixed cat corneas with 0.6-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and “bubbles” along the micromachined lines (clear, horizontal lines within stromal tissue). (B, D) BF images of the corneal region in (A, C) illustrating the visibility of spots of tissue destruction (arrow) and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A, C).
Figure 2.
 
Femtosecond IRIS in lightly fixed cat lens cortex just around the tissue-breakdown threshold. (A) DIC image of lines created with 0.8-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and the “bubbles” created. (B) BF image of the lens region in (A) illustrating the visibility of the bubbles and dark spots of tissue destruction (arrow), and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A).
Figure 2.
 
Femtosecond IRIS in lightly fixed cat lens cortex just around the tissue-breakdown threshold. (A) DIC image of lines created with 0.8-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and the “bubbles” created. (B) BF image of the lens region in (A) illustrating the visibility of the bubbles and dark spots of tissue destruction (arrow), and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A).
Figure 3.
 
Femtosecond IRIS in lightly fixed cat corneal stroma below the tissue-breakdown threshold. (A, C) DIC images of periodic line gratings created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of pieces of two cat corneas. Note the absence of tissue destruction (no dark spots or bubbles). (A, inset) Magnified portion of the grating. (B, D) BF images of corneal regions shown in (A) and (C), including the rectangular area magnified in the inset in (A). Note the poor visibility of the micromachined gratings under transmitted, bright light microscopy, which contrasts with the high visibility of the burn spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 1) .
Figure 3.
 
Femtosecond IRIS in lightly fixed cat corneal stroma below the tissue-breakdown threshold. (A, C) DIC images of periodic line gratings created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of pieces of two cat corneas. Note the absence of tissue destruction (no dark spots or bubbles). (A, inset) Magnified portion of the grating. (B, D) BF images of corneal regions shown in (A) and (C), including the rectangular area magnified in the inset in (A). Note the poor visibility of the micromachined gratings under transmitted, bright light microscopy, which contrasts with the high visibility of the burn spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 1) .
Figure 4.
 
Femtosecond IRIS in lightly fixed cat lens cortex below the tissue-breakdown threshold. (A) DIC image of a periodic line grating created with 0.5-nJ pulses and a scanning speed of 1 μm/s. Note the absence of tissue destruction and bubbles. (B) BF image of the lens region shown in (A). Note the poor visibility of the micromachined grating under transmitted, bright light microscopy, which contrasts with the high visibility of the dark spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 2) .
Figure 4.
 
Femtosecond IRIS in lightly fixed cat lens cortex below the tissue-breakdown threshold. (A) DIC image of a periodic line grating created with 0.5-nJ pulses and a scanning speed of 1 μm/s. Note the absence of tissue destruction and bubbles. (B) BF image of the lens region shown in (A). Note the poor visibility of the micromachined grating under transmitted, bright light microscopy, which contrasts with the high visibility of the dark spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 2) .
Figure 5.
 
Measuring refractive index change in IRIS-treated corneas immediately after treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the piece of cat cornea illustrated in Figure 3 . Note that some of the lines appear sharper than others, because the whole grating was not on the same focal plane when imaged under the microscope. (B) Graph plotting the second- and third-order diffraction efficiencies (×10−3) and the corresponding laser-induced refractive index changes of eight gratings micromachined in different corneal samples immediately after they were created. Inset: a photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8 nm He-Ne laser.
Figure 5.
 
Measuring refractive index change in IRIS-treated corneas immediately after treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the piece of cat cornea illustrated in Figure 3 . Note that some of the lines appear sharper than others, because the whole grating was not on the same focal plane when imaged under the microscope. (B) Graph plotting the second- and third-order diffraction efficiencies (×10−3) and the corresponding laser-induced refractive index changes of eight gratings micromachined in different corneal samples immediately after they were created. Inset: a photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8 nm He-Ne laser.
Figure 6.
 
Measuring refractive index change in IRIS-treated cat lenses immediately after the treatment. (A) DIC image of a periodic line grating created using 0.5-nJ pulses and a scanning speed of 1 μm/s in the cortex of the piece of cat lens illustrated in Figure 4 . (B) Graph plotting the first- and second-order diffraction efficiencies and the corresponding laser-induced refractive index changes of eight gratings micromachined in eight different lens cortices immediately after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 6.
 
Measuring refractive index change in IRIS-treated cat lenses immediately after the treatment. (A) DIC image of a periodic line grating created using 0.5-nJ pulses and a scanning speed of 1 μm/s in the cortex of the piece of cat lens illustrated in Figure 4 . (B) Graph plotting the first- and second-order diffraction efficiencies and the corresponding laser-induced refractive index changes of eight gratings micromachined in eight different lens cortices immediately after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 7.
 
Measuring refractive index change in IRIS-treated corneas 1 month after the treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the cat corneal piece shown in Figures 3A and 3B . After 1 month, the grating is still visible, but the clarity of the grating lines is markedly decreased, a likely result of corneal swelling and opacification. (B) Graph plotting the diffraction efficiencies (−10−3) and the corresponding refractive index changes of eight gratings measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 7.
 
Measuring refractive index change in IRIS-treated corneas 1 month after the treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the cat corneal piece shown in Figures 3A and 3B . After 1 month, the grating is still visible, but the clarity of the grating lines is markedly decreased, a likely result of corneal swelling and opacification. (B) Graph plotting the diffraction efficiencies (−10−3) and the corresponding refractive index changes of eight gratings measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 8.
 
Measuring refractive index change in IRIS-treated cat lenses 1 month after treatment. (A) DIC image of a periodic line grating created in lens cortex shown in Figure 4after 1 month of storage shows that the grating’s appearance did change significantly from its presentation in Figure 4 . (B) Graph plotting the diffraction efficiencies and the corresponding refractive index changes of eight gratings in eight lens cortex pieces measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 8.
 
Measuring refractive index change in IRIS-treated cat lenses 1 month after treatment. (A) DIC image of a periodic line grating created in lens cortex shown in Figure 4after 1 month of storage shows that the grating’s appearance did change significantly from its presentation in Figure 4 . (B) Graph plotting the diffraction efficiencies and the corresponding refractive index changes of eight gratings in eight lens cortex pieces measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
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Figure 1.
 
Femtosecond IRIS in lightly fixed cat corneal stroma just around the tissue-breakdown threshold. (A, C) DIC images of lines created in the stroma of two lightly fixed cat corneas with 0.6-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and “bubbles” along the micromachined lines (clear, horizontal lines within stromal tissue). (B, D) BF images of the corneal region in (A, C) illustrating the visibility of spots of tissue destruction (arrow) and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A, C).
Figure 1.
 
Femtosecond IRIS in lightly fixed cat corneal stroma just around the tissue-breakdown threshold. (A, C) DIC images of lines created in the stroma of two lightly fixed cat corneas with 0.6-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and “bubbles” along the micromachined lines (clear, horizontal lines within stromal tissue). (B, D) BF images of the corneal region in (A, C) illustrating the visibility of spots of tissue destruction (arrow) and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A, C).
Figure 2.
 
Femtosecond IRIS in lightly fixed cat lens cortex just around the tissue-breakdown threshold. (A) DIC image of lines created with 0.8-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and the “bubbles” created. (B) BF image of the lens region in (A) illustrating the visibility of the bubbles and dark spots of tissue destruction (arrow), and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A).
Figure 2.
 
Femtosecond IRIS in lightly fixed cat lens cortex just around the tissue-breakdown threshold. (A) DIC image of lines created with 0.8-nJ pulses and a scanning speed of 10 μm/s, showing dark spots of tissue destruction (arrow) and the “bubbles” created. (B) BF image of the lens region in (A) illustrating the visibility of the bubbles and dark spots of tissue destruction (arrow), and the relative invisibility of the rest of the lines that were clearly seen with DIC optics (A).
Figure 3.
 
Femtosecond IRIS in lightly fixed cat corneal stroma below the tissue-breakdown threshold. (A, C) DIC images of periodic line gratings created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of pieces of two cat corneas. Note the absence of tissue destruction (no dark spots or bubbles). (A, inset) Magnified portion of the grating. (B, D) BF images of corneal regions shown in (A) and (C), including the rectangular area magnified in the inset in (A). Note the poor visibility of the micromachined gratings under transmitted, bright light microscopy, which contrasts with the high visibility of the burn spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 1) .
Figure 3.
 
Femtosecond IRIS in lightly fixed cat corneal stroma below the tissue-breakdown threshold. (A, C) DIC images of periodic line gratings created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of pieces of two cat corneas. Note the absence of tissue destruction (no dark spots or bubbles). (A, inset) Magnified portion of the grating. (B, D) BF images of corneal regions shown in (A) and (C), including the rectangular area magnified in the inset in (A). Note the poor visibility of the micromachined gratings under transmitted, bright light microscopy, which contrasts with the high visibility of the burn spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 1) .
Figure 4.
 
Femtosecond IRIS in lightly fixed cat lens cortex below the tissue-breakdown threshold. (A) DIC image of a periodic line grating created with 0.5-nJ pulses and a scanning speed of 1 μm/s. Note the absence of tissue destruction and bubbles. (B) BF image of the lens region shown in (A). Note the poor visibility of the micromachined grating under transmitted, bright light microscopy, which contrasts with the high visibility of the dark spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 2) .
Figure 4.
 
Femtosecond IRIS in lightly fixed cat lens cortex below the tissue-breakdown threshold. (A) DIC image of a periodic line grating created with 0.5-nJ pulses and a scanning speed of 1 μm/s. Note the absence of tissue destruction and bubbles. (B) BF image of the lens region shown in (A). Note the poor visibility of the micromachined grating under transmitted, bright light microscopy, which contrasts with the high visibility of the dark spots and bubbles created when laser power above the tissue-breakdown threshold was used (Fig. 2) .
Figure 5.
 
Measuring refractive index change in IRIS-treated corneas immediately after treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the piece of cat cornea illustrated in Figure 3 . Note that some of the lines appear sharper than others, because the whole grating was not on the same focal plane when imaged under the microscope. (B) Graph plotting the second- and third-order diffraction efficiencies (×10−3) and the corresponding laser-induced refractive index changes of eight gratings micromachined in different corneal samples immediately after they were created. Inset: a photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8 nm He-Ne laser.
Figure 5.
 
Measuring refractive index change in IRIS-treated corneas immediately after treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the piece of cat cornea illustrated in Figure 3 . Note that some of the lines appear sharper than others, because the whole grating was not on the same focal plane when imaged under the microscope. (B) Graph plotting the second- and third-order diffraction efficiencies (×10−3) and the corresponding laser-induced refractive index changes of eight gratings micromachined in different corneal samples immediately after they were created. Inset: a photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8 nm He-Ne laser.
Figure 6.
 
Measuring refractive index change in IRIS-treated cat lenses immediately after the treatment. (A) DIC image of a periodic line grating created using 0.5-nJ pulses and a scanning speed of 1 μm/s in the cortex of the piece of cat lens illustrated in Figure 4 . (B) Graph plotting the first- and second-order diffraction efficiencies and the corresponding laser-induced refractive index changes of eight gratings micromachined in eight different lens cortices immediately after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 6.
 
Measuring refractive index change in IRIS-treated cat lenses immediately after the treatment. (A) DIC image of a periodic line grating created using 0.5-nJ pulses and a scanning speed of 1 μm/s in the cortex of the piece of cat lens illustrated in Figure 4 . (B) Graph plotting the first- and second-order diffraction efficiencies and the corresponding laser-induced refractive index changes of eight gratings micromachined in eight different lens cortices immediately after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 7.
 
Measuring refractive index change in IRIS-treated corneas 1 month after the treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the cat corneal piece shown in Figures 3A and 3B . After 1 month, the grating is still visible, but the clarity of the grating lines is markedly decreased, a likely result of corneal swelling and opacification. (B) Graph plotting the diffraction efficiencies (−10−3) and the corresponding refractive index changes of eight gratings measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 7.
 
Measuring refractive index change in IRIS-treated corneas 1 month after the treatment. (A) DIC image of a periodic line grating created using 0.3-nJ pulses and a scanning speed of 0.7 μm/s in the stromal layer of the cat corneal piece shown in Figures 3A and 3B . After 1 month, the grating is still visible, but the clarity of the grating lines is markedly decreased, a likely result of corneal swelling and opacification. (B) Graph plotting the diffraction efficiencies (−10−3) and the corresponding refractive index changes of eight gratings measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 8.
 
Measuring refractive index change in IRIS-treated cat lenses 1 month after treatment. (A) DIC image of a periodic line grating created in lens cortex shown in Figure 4after 1 month of storage shows that the grating’s appearance did change significantly from its presentation in Figure 4 . (B) Graph plotting the diffraction efficiencies and the corresponding refractive index changes of eight gratings in eight lens cortex pieces measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
Figure 8.
 
Measuring refractive index change in IRIS-treated cat lenses 1 month after treatment. (A) DIC image of a periodic line grating created in lens cortex shown in Figure 4after 1 month of storage shows that the grating’s appearance did change significantly from its presentation in Figure 4 . (B) Graph plotting the diffraction efficiencies and the corresponding refractive index changes of eight gratings in eight lens cortex pieces measured 1 month after they were created. Inset: photograph of the diffraction pattern obtained when illuminating the grating shown in (A) with a 632.8-nm He-Ne laser.
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