Temporal evolution of the biological response to laser-induced refractive index change (LIRIC) in rabbit corneas.

Laser-induced refractive index change (LIRIC) is a new, non-incisional, non-ablative, femtosecond photo-modification technique being developed for vision correction in humans. Prior, exvivo studies showed intra-tissue refractive index change to induce minimal cell death, restricted to the laser focal zone in the corneal stroma, and with no observable damage to the epithelium or endothelium. Here, we used live rabbits to ascertain longer-term consequences of LIRIC in vivo. Specifically, we assessed cell death, fibrosis, corneal nerve distribution, endothelial cell density, and corneal structure for up to 3 months after LIRIC. A +2.5 D gradient-index LIRIC Fresnel lens was inscribed inside 20 applanated corneas of Dutch Belted rabbits, over a circular region of the mid-stroma measuring 4.5 mm in diameter. Twelve additional rabbit eyes were used as applanation-only controls to differentiate the effects of laser treatment and suction applanation on biological and structural parameters. In vivo optical measurements were performed pre-operatively, then immediately, 2, 4, and 12 weeks after the procedure, to measure endothelial cell density and changes in corneal structure. Groups of four rabbits were sacrificed at 4 hours, 2, 4, and 12 weeks after LIRIC for histological determinations; the TUNEL assay was used to evaluate cell death, H&E staining was used to assess inflammatory infiltration, and immunostaining for α-smooth muscle actin (α-SMA) and ßIII tubulin (Tuj-1) was performed to assess myofibroblast differentiation and corneal nerve distribution, respectively. Consistent with prior ex vivo data, only minimal cell death was observed in the laser focal zone, with TUNEL-positive cells restricted to the stromal region of refractive index change 4 h after LIRIC. No TUNEL-positive cells were evident anywhere in the cornea 2, 4, or 12 weeks after LIRIC. Applanation-only corneas were completely TUNEL-negative. Neither LIRIC-treated nor applanation-only eyes exhibited α-SMA-positive staining or altered corneal nerve distributions at any of the time points examined. In vivo confocal imaging revealed normal endothelial cell densities in all eyes (whether LIRIC-treated or applanation-only) at all time points. Optical coherence tomography showed suction applanation to cause a temporary decrease in central corneal thickness, which returned to normal within 4 h. Corneas into which LIRIC Fresnel lenses were written while applanated did not undergo major structural or shape changes beyond the temporary thinning already described for suction applanation. The present findings suggest that LIRIC patterns, which generated a clinically-relevant refractive correction in the mid-stromal region of live rabbit corneas, induced little-to-no disruption to corneal structure and biology for 3 months after the procedure. This affirms the relative safety of LIRIC and predicts that compared to traditional laser vision correction surgeries, common post-operative complications such as dry eye, haze, or patient discomfort may be entirely avoided.

Modification of surface morphology of hydrogels due to subsurface femtosecond laser micromachining.

In this paper, we studied the effects of subsurface femtosecond laser micromachining on surface morphology in hydrogels. Depending on material properties and writing conditions, we found surface bumps when materials were hydrated, and trenches when they were dehydrated, which can be attributed to the localized change in water concentration. Such wavy surfaces by laser-induced refractive index change are not desirable in clinical contact lenses. Therefore, the minimization of surface bumps is necessary to ensure the user eye wearing comfort. In addition, we examined the optical effects of the surface features using interferometry and the surface morphology using profilometry. Finally, we proposed a simplified mechanical model based on localized swelling.

Increase in efficacy of near-infrared femtosecond micromachining in ophthalmic hydrogels with the addition of sodium fluorescein, rose bengal, and riboflavin.

We report on the effect of exogenous doping agents with large two-photon absorption cross sections on the efficacy of near-infrared femtosecond micromachining in ophthalmic hydrogels. Contaflex GM Advance 58 hydrogels were solution doped in low concentrations of sodium fluorescein, rose bengal, and riboflavin dissolved in balanced salt solution prior to femtosecond micromachining with three near-infrared wavelengths: 720, 800, and 1035 nm. Using any of the three doping agents in the concentrations studied produced an increase in the amount of optical phase change induced in the material at all writing wavelengths and reduced the amount of power needed to induce a desired amount of phase change.

Contrasting cellular damage after Blue-IRIS and Femto-LASIK in cat cornea.

Blue-intra-tissue refractive index shaping (Blue-IRIS) is a new approach to laser refractive correction of optical aberrations in the eye, which alters the refractive index of the cornea rather than changing its shape. Before it can be implemented in humans, it is critical to establish whether and to what extent, Blue-IRIS damages the cornea. Here, we contrasted the impact of -1.5 D cylinder refractive corrections inscribed using either Blue-IRIS or femtosecond laser in-situ keratomileusis (femto-LASIK) on corneal cell viability. Blue-IRIS was used to write a -1.5 D cylinder gradient index (GRIN) lens over a 2.5 mm by 2.5 mm area into the mid-stromal region of the cornea in six freshly-enucleated feline eyes. The same correction (-1.5 D cylinder) was inscribed into another four cat eyes using femto-LASIK. Six hours later, all corneas were processed for histology and stained for terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and p-γ-H2AX to label damaged cells. In Blue-IRIS-treated corneas, no tissue was removed and TUNEL-stained cells were confined to the laser focal zone in the stroma. In femto-LASIK, photoablation removed 14 µm of anterior stroma, but in addition, TUNEL-positive cells clustered across the femto-flap, the epithelium at the flap edges and the stroma below the ablation zone. Keratocytes positive for p-γ-H2AX were seen adjacent to all Blue-IRIS focal zones, but were completely absent from femto-LASIK-treated corneas. Unlike femto-LASIK, Blue-IRIS attains refractive correction in the cornea without tissue removal and only causes minimal, localized keratocyte death within the laser focal zones. In addition, Blue-IRIS induced DNA modifications associated with phosphorylation of γ-H2AX in keratocytes adjacent to the laser focal zones. We posit that this p-γ-H2AX response is related to alterations in chromatin structure caused by localized changes in osmolarity, a possible mechanism for the induced refractive index changes.

Speckle-free femtosecond red-green-blue (RGB) source from a fiber laser driven spectrally efficient two zero dispersion wavelength fiber source.

We report a novel ultrafast red-green-blue (RGB) laser source based on second harmonic generation from a two zero dispersion wavelength (TZDW) fiber continuum source. The TZDW fiber source consists of a custom-built Yb:fiber amplifier and a commercially available TZDW photonic crystal fiber (PCF) which enables low noise and efficient frequency conversion from the 1035 nm pump source to two spectrally localized pulses centered at 850 nm and 1260 nm with 39.6% and 33.7% power efficiencies. With angularly multiplexed simultaneous phase matching, we achieve mW average power of red, green and blue pulses at 630 nm, 517 nm and 426 nm from single pass second harmonic generation. With broad RGB bandwidths of 7.4 nm, 3.2 nm and 5.2 nm, the source is inherently speckle-free while maintaining an excellent color rendering capability with higher than 99.7% excitation purity of the RGB color primaries, leading to the coverage of 192% NTSC color gamut (CIE 1976). The reported source features a simple system geometry; its potential in power scaling is discussed with currently available technologies.

Yb:fiber laser-based, spectrally coherent and efficient generation of femtosecond 1.3-µm pulses from a fiber with two zero-dispersion wavelengths.

We report, to the best of our knowledge, the first experimental characterization of spectral coherence properties of wavelength conversion inside photonic crystal fibers with two zero-dispersion wavelengths (TZDWs) and demonstrate a low-noise femtosecond 1.3-µm source employing the TZDW fiber and a 1.3-W, 240-fs Yb:fiber amplifier as the seeding source. Theoretical investigation shows that pulse evolution in our TZDW fiber source is dominated by parametric amplification seeded by self-phase modulation broadening which efficiently converts the pump energy into two new wavelength bands in a deterministic manner, leading to low noise and coherent excitation of femtosecond pulses tunable in the 1.3-µm spectral region, with up to 3 nJ of pulse energy at 32% of conversion efficiency.

Broadly tunable femtosecond mid-infrared source based on dual photonic crystal fibers.

We report a novel scheme of generating broadly tunable femtosecond mid-IR pulses based on difference frequency mixing the outputs from dual photonic crystal fibers (PCF). With a 1.3 W, 1035 nm, 300 fs and 40 MHz Yb fiber chirped pulse amplifier as the laser source, a PCF with single zero dispersion wavelength (ZDW) at the laser wavelength is employed to spectrally broaden a portion of the laser pulses. Facilitated by self-phase modulation, its output spectrum possesses two dominant outermost peaks that can be extended to 970 nm and 1092 nm. A different PCF with two closely spaced ZDWs around the laser wavelength is used to generate the intense Stokes pulses between 1240 - 1260 nm. Frequency mixing the dual PCFs outputs in an AgGaS(2) crystal results in mid-IR pulses broadly tunable from 4.2 µm to 9 µm with a maximum average power of 640 µW at 4.5 µm, corresponding to 16 pJ of pulse energy.

Difference frequency generation of femtosecond mid infrared pulses employing intense Stokes pulses excitation in a photonic crystal fiber.

We demonstrate a novel method of generating milli-watt level mid-IR (MIR) pulses based on difference frequency mixing of the output from a 40 MHz Yb fiber Chirped Pulse Amplifier (CPA) and the intense Stokes pulses generated in a photonic crystal fiber (PCF) with two closely spaced zero dispersion wavelengths (ZDW). By taking advantage of the unique dispersion profile of the fiber, high power narrowband Stokes pulses are selectively generated in the normal dispersion region of the PCF with up to 1.45 nJ of pulse energy. Mixing with 12 nJ of pump pulses at 1035 nm in a type-II AgGaS(2) crystal yields MIR pulses around 5.5 µm wavelength with up to 3 mW of average power and 75 pJ of pulse energy. The reported method can be extended to generation of other MIR wavelengths by selecting PCFs with different second ZDWs or engineering the fiber dispersion profile via longitudinal tapering.

Blue-LIRIC in the rabbit cornea: efficacy, tissue effects, and repetition rate scaling.

Laser-induced refractive index change (LIRIC) is being developed as a non-invasive way to alter optical properties of transparent, ophthalmic materials including corneas ex vivo and in vivo. This study examined the optical and biological effects of blue-LIRIC (wavelengths 400-405 nm) of ex-vivo rabbit corneas. Following LIRIC treatment at low and high repetition rates (8.3 MHz and 80 MHz, respectively), we interferometrically measured optical phase change, obtained transmission electron microscopy (TEM) micrographs, and stained histological sections with collagen hybridizing peptides (CHP) to assess the structural and organizational changes caused by LIRIC at different repetition rates. Finally, we performed power and scan speed scaling experiments at three different repetition rates (1 MHz, 8.3 MHz, and 80 MHz) to study their impact on LIRIC efficacy. Histologic co-localization of CHP and LIRIC-generated green autofluorescence signals suggested that collagen denaturation had occurred in the laser-irradiated region. TEM imaging showed different ultrastructural modifications for low and high repetition rate writing, with discrete homogenization of collagen fibrils at 80 MHz, as opposed to contiguous homogenization at 8.3 MHz. Overall, this study confirmed that LIRIC efficacy can be dramatically increased, while still avoiding tissue ablation, by lowering the repetition rate from 80 MHz to 8.3 MHz. Modeling suggests that this is due to a higher, single-pulse, energy density deposition at given laser powers during 8.3 MHz LIRIC.

Silicon Nanomembrane Filtration and Imaging for the Evaluation of Microplastic Entrainment along a Municipal Water Delivery Route.

To better understand the origin of microplastics in municipal drinking water, we evaluated 50 mL water samples from different stages of the City of Rochester's drinking water production and transport route, from Hemlock Lake to the University of Rochester. We directly filtered samples using silicon nitride nanomembrane filters with precisely patterned slit-shaped pores, capturing many of the smallest particulates (<20 µm) that could be absorbed by the human body. We employed machine learning algorithms to quantify the shapes and quantity of debris at different stages of the water transport process, while automatically segregating out fibrous structures from particulate. Particulate concentrations ranged from 13 to 720 particles/mL at different stages of the water transport process and fibrous pollution ranged from 0.4 to 8.3 fibers/mL. A subset of the debris (0.2-8.6%) stained positively with Nile red dye which identifies them as hydrophobic polymers. Further spectroscopic analysis also indicated the presence of many non-plastic particulates, including rust, silicates, and calcium scale. While water leaving the Hemlock Lake facility is mostly devoid of debris, transport through many miles of piping results in the entrainment of a significant amount of debris, including plastics, although in-route reservoirs and end-stage filtration serve to reduce these concentrations.

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

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.

Low loss fusion splicing of micron scale silica fibers.

Tapered micron-sized optical fibers may be important in the future for development of microscale integrated photonic devices. Complex photonic circuits require many devices and a robust technique for interconnection. We demonstrate splicing of four micron diameter step-index air-clad silica microfibers using a CO2 laser. We obtain splice losses lower than 0.3%. Compared with evanescent coupling of microfibers, our splices are more mechanically stable and efficient.

Large enhancement of femtosecond laser micromachining speed in dye-doped hydrogel polymers.

Ophthalmologic hydrogel polymers are doped with Fluorescein or Coumarin dyes prior to the femtosecond laser micromachining process. We find that the achievable micromachining writing speed can be greatly increased while maintaining large refractive index changes (up to +0.08). Compared with previous results in dye-doped polymers that do not contain water such as PMMA, we obtain much larger index changes and much faster writing speeds.

End-sealing short dispersion micromanaged tapered holey fibers by hole-collapsing.

We have recently shown that fiber dispersion can be manipulated on a sub-millimeter scale, and discussed its importance in production of low-noise supercontinuum generation. In this paper, we report the fabrication of dispersion micromanaged (DMM) holey fibers that have been structurally modified to offer greater environmental stability and have reduced sensitivity towards alignment in input coupling. Our results show that end-sealed devices can be made while retaining key features of the dispersion micromanagement.

Self referenced Yb-fiber-laser frequency comb using a dispersion micromanaged tapered holey fiber.

We demonstrate a fully stabilized frequency comb in the 1mum spectral region based on an Yb-fiber oscillator and a cladding pumped chirped pulse Yb-fiber amplifier whose output is spectrally broadened in a dispersion micromanaged holey fiber. The dispersion micromanaged fiber is used to generate efficient, low noise spectral components at 523nm which are heterodyned with the second harmonic of the amplifier output for standard f-to-2f self-referenced carrier envelope offset frequency detection. For comb stabilization we phase-lock this offset frequency and the oscillator repetition frequency simultaneously to an RF reference by feedback controlling the oscillator pump diode current and the driving voltage of an intracavity piezo-electric fiber stretcher respectively.

Comparable change in stromal refractive index of cat and human corneas following blue-IRIS.

Blue intratissue refractive index shaping (blue-IRIS) is a method with potential to correct ocular refraction noninvasively in humans. To date, blue-IRIS has only ever been applied to cat corneas and hydrogels. To test the comparability of refractive index change achievable in cat and human tissues, we used blue-IRIS to write identical phase gratings in ex vivo feline and human corneas. Femtosecond pulses (400 nm) were focused ? 300 ?? ? m below the epithelial surface of excised cat and human corneas and scanned to write phase gratings with lines ? 1 ?? ? m wide, spaced 5 ?? ? m apart, using a scan speed of 5 ?? mm / s . Additional cat corneas were used to test writing at 3 and 7 ?? mm / s in order to document speed dependence of the refractive index change magnitude. The first-order diffraction efficiency was immediately measured and used to calculate the refractive index change attained. Our data show that blue-IRIS induces comparable refractive index changes in feline and human corneas, an essential requirement for further developing its use as a clinical vision correction technique.

Large refractive index change in silicone-based and non-silicone-based hydrogel polymers induced by femtosecond laser micro-machining.

A Ti:Sapphire femtosecond laser with a pulse energy of 1.3 nJ at a 93 MHz repetition rate has been used to micro-machine optical gratings inside several silicone-based and non-silicone-based hydrogel polymers. By measuring the diffraction efficiency of the gratings at 632.8 nm, we find as large as 0.06+/- 0.005 average refractive index change within the irradiated area.

First demonstration of ocular refractive change using blue-IRIS in live cats.

PURPOSE: To determine the efficacy of intratissue refractive index shaping (IRIS) using 400-nm femtosecond laser pulses (blue light) for writing refractive structures directly into live cat corneas in vivo, and to assess the longevity of these structures in the eyes of living cats. METHODS: Four eyes from two adult cats underwent Blue-IRIS. Light at 400 nm with 100-femtosecond (fs) pulses were tightly focused into the corneal stroma of each eye at an 80-MHz repetition rate. These pulses locally increased the refractive index of the corneal stroma via an endogenous, two-photon absorption process and were used to inscribe three-layered, gradient index patterns into the cat corneas. The optical effects of the patterns were then tracked using optical coherence tomography (OCT) and Shack-Hartmann wavefront sensing. RESULTS: Blue-IRIS patterns locally changed ocular cylinder by -1.4 ± 0.3 diopters (D), defocus by -2.0 ± 0.5 D, and higher-order root mean square (HORMS) by 0.31 ± 0.04 µm at 1 month post-IRIS, without significant changes in corneal thickness or curvature. Refractive changes were maintained for the duration they were tracked, 12 months post-IRIS in one eye, and just more than 3 months in the remaining three eyes. CONCLUSIONS: Blue-IRIS can be used to inscribe refractive structures into live cat cornea in vivo that are stable for at least 12 months, and are not associated with significant alterations in corneal thicknesses or radii of curvature. This result is a critical step toward establishing Blue-IRIS as a promising technique for noninvasive vision correction.

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

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.

High-resolution, noninvasive, two-photon fluorescence measurement of molecular concentrations in corneal tissue.

PURPOSE: To perform high-resolution, noninvasive, calibrated measurements of the concentrations and diffusion profiles of fluorescent molecules in the live cornea after topical application to the ocular surface. METHODS: An 800-nm femtosecond laser was used to perform two-photon fluorescence (TPF) axial scanning measurements. Calibration solutions consisting of sodium fluorescein (Na-Fl; concentration range, 0.01%-2.5%) and riboflavin (concentration range, 0.0125%-0.1%) were tested in well slides, and TPF signals were assessed. Excised feline eyeballs preserved in corneal storage medium and with either intact or removed corneal epithelia were then treated with Na-Fl, riboflavin, or fluorescein dextran (Fl-d) of different molecular weight (MW) for 30 minutes. Calibrated TPF was then used immediately to measure the concentration of these molecules across the central corneal depth. RESULTS: The axial resolution of our TPF system was 6 µm, and a linear relationship was observed between TPF signal and low concentrations of most fluorophores. Intact corneas treated with Na-Fl or riboflavin exhibited a detectable penetration depth of only approximately 20 µm, compared with approximately 400 to 600 µm when the epithelium was removed before fluorophore application. Peak concentrations for intact corneas were half those attained with epithelial removal. Debrided corneas treated with 2,000,000 MW Fl-d showed a half-maximum penetration depth of 156.7 µm compared with 384 µm for the 3,000 MW dextran. The peak concentration of the high MW dextran was one quarter that of the lower MW dextran. CONCLUSIONS: TPF is an effective, high-resolution, noninvasive method of quantifying the diffusion and concentration of fluorescent molecules across the cornea.