Retinal safety evaluation of two-photon laser scanning in rats.

Safe use of retinal imaging with two-photon excitation in human eyes is crucial, as the effects of ultrashort pulsed lasers on the retina are relatively unknown. At the time of the study, the laser safety standards were inadequate due to the lack of biological data. This article addresses the feasibility of two-photon retinal imaging with respect to laser safety. In this study, rat retinas were evaluated at various laser exposure levels and with different laser parameters to determine the effects of laser-induced optical damage. The results were experimentally verified using confocal reflectance imaging, two-photon fluorescein angiography, immunohistochemistry, and correlated to the IEC 60825-1 laser safety standard.

In vivo two-photon imaging of retina in rabbits and rats.

The purpose of this study was to evaluate the retina using near-infrared (NIR) two-photon scanning laser ophthalmoscopy. New Zealand white rabbits, albino rats, and brown Norway rats were used in this study. An autofluorescence image of the retina, including the retinal cells and its associated vasculatures was obtained by a real-time scan using the ophthalmoscope. Furthermore, the retinal vessels, nerve fiber layers and the non-pigmented retina were recorded with two-photon fluorescein angiography (FA); and the choroidal vasculatures were recorded using two-photon indocyanine green angiography (ICGA). Two-photon ICGA was achieved by exciting a second singlet state at ∼398 nm. Simultaneous two-photon FA and two-photon ICGA were performed to characterize the retinal and choroidal vessels with a single injection. The minimum laser power threshold required to elicit two-photon fluorescence was determined. The two-photon ophthalmoscope could serve as a promising tool to detect and monitor the disease progression in animal models. Moreover, these high-resolution images of retinal and choroidal vessels can be acquired in a real-time scan with a single light source, requiring no additional filters for FA or ICGA. The combination of FA and ICGA using the two-photon ophthalmoscope will help researchers to characterize the retinal diseases in animal models, and also to classify the types (classic, occult or mixed) of choroidal neovascularization (CNV) in macular degeneration. Furthermore, the prototype can be adapted to image the retina of rodents and rabbits.

Chemical basis for alteration of an intraocular lens using a femtosecond laser.

The chemical basis for the alteration of the refractive properties of an intraocular lens with a femtosecond laser was investigated. Three different microscope setups have been used for the study: Laser Induced Fluorescence (LIF) microscopy, Raman microscopy and coherent anti-Stokes Raman Scattering (CARS) microscopy. Photo-induced hydrolysis of polymeric material in aqueous media produces two hydrophilic functional groups: acid group and alcohol group. The spectral signatures identify two of the hydrophilic polar molecules as N-phenyl-4-(phenylazo)-benzenamine (C18H15N3) and phenazine-1-carboxylic acid (C13H8N2O2). The change in hydrophilicity results in a negative refractive index change in the laser-treated areas.

Creation of a refractive lens within an existing intraocular lens using a femtosecond laser.

PURPOSE: To assess the efficiency and effectiveness of the technology that creates a hydrophilicity-based refractive index change within a standard intraocular lens (IOL) to alter the refractive characteristics of the IOL. SETTING: Perfect Lens LLC, Irvine, California, USA. DESIGN: Experimental study. METHODS: The IOL used in this experiment was a standard hydrophobic model (EC-1Y). The refractive index of the material was changed by exposure of the material to a femtosecond laser and the subsequent absorption of water by the material. An experimental system using a femtosecond laser, an acoustic-optic modulator, beam-shaping optics, a scan system, and an objective lens was used to create the refractive index change within the IOL. Experiments were performed to determine the optimum wavelength, energy per pulse, and line spacing to produce the refractive index shaping lens. A power and modulation transfer function (MTF) measurement device for refractive and diffractive IOLs was used to measure the diopter and MTF before and after the creation of the refractive index shaping lens. RESULTS: The technology successfully altered the refractive characteristics of the IOL. The refractive index change altered the diopter (D) of the IOL (to within ±0.1 D of the targeted change) without significant diminution in the MTF (<0.1 or MTF ≥0.51 for the 100 lp/mm measurement). CONCLUSION: The refractive properties of an IOL can be altered by building a refractive index shaping lens within an IOL using a femtosecond laser with minimal diminution in MTF. FINANCIAL DISCLOSURE: All authors are employed by Perfect Lens, LLC and have a financial interest in the products of Perfect Lens, LLC.

In vivo non-linear optical (NLO) imaging in live rabbit eyes using the Heidelberg Two-Photon Laser Ophthalmoscope.

Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW fs laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 mum diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 h after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas.

High-speed two-photon excited autofluorescence imaging of ex vivo human retinal pigment epithelial cells toward age-related macular degeneration diagnostic.

Age-related macular degeneration (AMD) is among the major concerns in ophthalmology, as it is the primary cause for irreversible blindness in developed countries. Nevertheless, there is poor understanding of the origins and mechanisms that trigger this important ocular disease. In common clinical pratice, AMD is monitored by autofluorescence imaging of the retinal pigment epithelial (RPE) cells through a confocal scanning laser ophthalmoscope. The RPE cells derive their dominant autofluorescence from the lipofuscin granules that accumulate in the cytoplasm with increasing age and disease. We explored a different approach to retinal RPE imaging using two-photon excited autofluorescence, offering intrinsic three-dimensional resolution, larger sensing depth and reduced photodamage compared to single-photon excited fluorescence ophthalmoscopy. A two-photon microscope, based on the architecture of a conventional scanning laser ophthalmoscope (HRT, Heidelberg Engineering, Germany), was designed for autofluorescence imaging on retina samples from postmortem human-donor eyes. We were able to visualize at video-rate speed single RPE lipofuscin granules, demonstrating the potential to develop this method toward clinical practice for patients with RPE-related retinal disease like AMD.

Femtosecond laser corneal ablation threshold: dependence on tissue depth and laser pulse width.

BACKGROUND AND OBJECTIVE: Diode pumped, all-solid-state ultrafast lasers are now widely used to perform minimally invasive refractive surgery and keratoplasty procedures. Despite such use, a systematic study concerning ultrafast laser-tissue interactions is lacking. We determined the corneal ablation threshold as a function of the laser pulse width and stromal depth by simultaneous monitoring of the intensity of the laser-induced plasma and the second harmonic generation signals (SHG) from the collagen. STUDY DESIGN/MATERIALS AND METHODS: Ablation thresholds in porcine cornea samples were determined using three diode pumped all-solid-state ultrafast lasers (a Nd:glass femtosecond laser, a Yb:KYW femtosecond laser, and a Nd: YAG picosecond laser) over a range from 800 femtoseconds to 20 picoseconds. RESULTS: Corneal ablation threshold remained nearly constant within the first 200 microm of stroma and was consistent with previous findings with the threshold proportional to the square root of the laser pulse width. CONCLUSION: Corneal ablation thresholds can be precisely determined by simultaneous monitoring of the intensity of the laser-induced plasma and the SHG from the cornea.

Age-related structural abnormalities in the human retina-choroid complex revealed by two-photon excited autofluorescence imaging.

The intensive metabolism of photoreceptors is delicately maintained by the retinal pigment epithelium (RPE) and the choroid. Dysfunction of either the RPE or choroid may lead to severe damage to the retina. Two-photon excited autofluorescence (TPEF) from endogenous fluorophores in the human retina provides a novel opportunity to reveal age-related structural abnormalities in the retina-choroid complex prior to apparent pathological manifestations of age-related retinal diseases. In the photoreceptor layer, the regularity of the macular photoreceptor mosaic is preserved during aging. In the RPE, enlarged lipofuscin granules demonstrate significantly blue-shifted autofluorescence, which coincides with the depletion of melanin pigments. Prominent fibrillar structures in elderly Bruch's membrane and choriocapillaries represent choroidal structure and permeability alterations. Requiring neither slicing nor labeling, TPEF imaging is an elegant and highly efficient tool to delineate the thick, fragile, and opaque retina-choroid complex, and may provide clues to the trigger events of age-related macular degeneration.

Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:Sapphire laser.

PURPOSE: To record the distribution and spectrum of human retinal pigment epithelial cell lipofuscin (LF) by two-photon-excited fluorescence (TPEF) and confocal laser scanning microscopy. METHODS: Ex vivo TPEF imaging of the human retinal pigment epithelium (RPE) of human donor eyes was conducted with a multiphoton laser scanning microscope that employs a femtosecond Ti:sapphire laser as an excitation laser source. The spectrum of autofluorescence of LF granules was analyzed with a confocal laser scanning microscope coupled to a UV argon laser. RESULTS: TPEF examination allowed for imaging of RPE cell morphology and intracellular distribution of LF granules with high-contrast and submicrometer resolution. Variations in cell size and shape as well as in autofluorescence spectra of individual LF granules were recorded. The typical diameter of LF granules was found to be below 1 mum, with some RPE cells possessing larger granules. Remarkably, enhanced blue-green autofluorescence was observed from these larger LF granules. CONCLUSIONS: TPEF imaging represents a novel tool for the investigation of morphologic and spectral characteristics of human RPE cells. Spectral variations of individual LF granules may indicate differences in the complex molecular composition. Compared to conventional single-photon excited autofluorescence, TPEF with a tunable laser source allows for reduced photo damage and deeper sensing depth. It may help to elucidate further the pathophysiological role of LF accumulation as a common downstream pathogenetic pathway in retinal diseases. With the proof of principle from this ex vivo study, further work is now planned to evaluate the safety of TPEF RPE imaging in RPE cultures and animal models.

Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells.

Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

Wavefront reconstruction with artificial neural networks.

In this work, a new approach, a method using artificial neural networks was applied to reconstruct the wavefront. First, the optimal structure of neural networks was found. Then, the networks were trained on both noise-free and noisy spot patterns. The results of the wavefront reconstruction with artificial neural networks were compared to those obtained through the least square fit and singular value decomposition method.

Second harmonic generation imaging of collagen fibrils in cornea and sclera.

Collagen, as the most abundant protein in the human body, determines the unique physiological and optical properties of the connective tissues including cornea and sclera. The ultrastructure of collagen, which conventionally can only be resolved by electron microscopy, now can be probed by optical second harmonic generation (SHG) imaging. SHG imaging revealed that corneal collagen fibrils are regularly packed as a polycrystalline lattice, accounting for the transparency of cornea. In contrast, scleral fibrils possess inhomogeneous, tubelike structures with thin hard shells, maintaining the high stiffness and elasticity of the sclera.

Second-harmonic imaging of cornea after intrastromal femtosecond laser ablation.

Nonlinear laser scanning microscopy is widely used for noninvasive imaging in cell biology and tissue physiology. However, multiphoton fluorescence imaging of dense, transparent connective tissue (e.g., cornea) is challenging since sophisticated labeling or slicing is necessary. High-resolution, high-contrast second harmonic generation (SHG) imaging of corneal tissue based on the intrinsic structure of collagen is discussed. The three-dimensional corneal ultrastructure in depths up to hundreds of microns can be probed noninvasively, without any staining or mechanical slicing. As an important application of second harmonic imaging in ophthalmology, the modification of corneal ultrastructure using femtosecond laser intrastromal ablation is systematically investigated to evaluate next-generation refractive surgical approaches.

Mini-invasive corneal surgery and imaging with femtosecond lasers.

Based on the transparency of corneal tissue and on laser plasma mediated non-thermal tissue ablation, near infrared femtosecond lasers are promising tools for minimally invasive intrastromal refractive surgery. Femtosecond lasers also enable novel nonlinear optical imaging methods like second harmonic corneal imaging. The microscopic effects of femtosecond laser intrastromal surgery were successfully visualized by using second harmonic corneal imaging with diffraction limited resolution, strong imaging contrast and large sensing depth, without requiring tissue fixation or sectioning. The performance of femtosecond laser intrastromal surgery proved to be precise, repeatable and predictable. It might be possible to integrate both surgical and probing functions into a single femtosecond laser system.

Very fast wave-front measurements at the human eye with a custom CMOS-based Hartmann-Shack sensor.

We describe what we believe to be the first wave-front measurements of the human eye at a sampling rate of 300 Hz with a custom Hartmann-Shack wave-front sensor that uses complementary metal-oxide semiconductor (CMOS) technology. This sensor has been developed to replace standard charge-coupled device (CCD) cameras and the slow software image processing that is normally used to reconstruct the wave front from the focal-plane image of a lenslet array. We describe the sensor's principle of operation and introduce the performance with static wave fronts. The system has been used to measure human-eye wave-front aberrations with a bandwidth of 300 Hz, which is approximately an order of magnitude faster than with standard software-based solutions. Finally, we discuss the measured data and consider further improvements to the system.