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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.

Adaptive optics has emerged as an empowering technology for retinal imaging with cellular resolution. This technology holds potential for non-invasive detection and diagnoses of the most common eye diseases such as glaucoma, diabetic retinopathy and age-related macular degeneration (AMD). However, the cost and complexity of adaptive optics ophthalmoscopes currently impede its clinical use. A scanning laser ophthalmoscope with phase plate has the potential to establish adaptive optics in a clinical application by simplifying its incorporation in the prevailing systems. Phase plates can correct aberrations down to diffraction limit and can be easily incorporated into ophthalmoscopes to improve the retinal image quality.

More than 25 years ago, the first in vivo measurement of the eye’s wave aberration was demonstrated at the University of Heidelberg, using the Shack-Hartmann method. The continuous measurement of ocular aberrations paved the way to employ adaptive optics for the correction of ocular aberrations in ophthalmoscopy. Various new diagnostic and treatment modalities have since been successfully introduced, e.g. adaptive optics scanning laser ophthalmoscopy (AO-SLO), adaptive optics optical coherence tomography (AO-OCT) and adaptive optics two-photon ophthalmoscopy (AO-TPO). Employing adaptive optics techniques, the limits of human vision can be assessed. With the development of wavefront-guided laser refractive surgery, the quest for optically perfect vision is being pursued over the last 20 years.

Cataract surgeons are in constant pursuit to achieve optimal visual outcomes for their patients. Therefore, numerous technologies and techniques have emerged, most of which are designed to assist the surgeon preoperatively and intraoperatively. The in vivo modification is achieved by creating a lens within an existing IOL using a femtosecond laser. The lens within the lens is created by altering the hydrophilicity of an existing and standard IOL in certain designated areas. The changes created by the perfect lens technology alter the IOL’s refractive outcome and enable the surgeon to create the patient’s desired vision in a simple postoperative in-office procedure.

Femtosecond-Laser-Assisted cataract surgery (FLACS) offers surgeons a reproducible, non-invasive technique to replace the least predictable and technically most demanding steps of conventional cataract procedures. A computer-guided laser linked to an optical imaging system (e.g. OCT) performs the corneal incision, capsulotomy, and lens fragmentation steps, thus changing the requirements associated with traditional techniques by removing the need for blade incisions and reducing phacoemulsification time and energy. Initial research has confirmed appropriate safety and efficacy of Femtosecond-Laser-Assisted cataract surgery, with improvements in anterior capsulotomy, phacofragmentation, and corneal incision. The potential gains in precision associated with FLACS may improve safety and clinical outcomes.

Since the invention of the first ophthalmoscope in 1851, the quest for quantitative in vivo imaging of ocular surfaces for diagnostic purposes was initiated. Confocal microscopy has been widely implemented in clinics providing excellent spatial resolution and enabling optical sectioning. With the introduction of ultrashort pulsed laser sources, multiphoton microscopy became feasible. Multiphoton microscopy allows 3D tissue imaging. Collagen, elastin, NAD(P)H etc., can be imaged by multiphoton microscopy, providing structural and functional information of the cornea. In this article, we will discuss the principles and potential use and limitations of confocal and multiphoton microscopy for corneal imaging.

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.

More than 25 years ago, the first in vivo measurement of the eye’s wave aberration was demonstrated at the University of Heidelberg, using the Shack-Hartmann method. Since then, various new diagnostic and treatment modalities have been successfully introduced, like adaptive optics scanning laser ophthalmoscopy (AO-SLO), adaptive optics optical coherence tomography (AO-OCT) and adaptive optics two-photon ophthalmoscopy (AO-TPO), as well as customized refractive-surgical techniques, like customized LASIK (e.g. CustomVue), femtosecond laser refractive surgery (e.g. ReLEx, SMILE), femtosecond laser assisted cataract surgery (FLACS) and various customized intraocular lens (IOL) developments, including postoperative in-vivo IOL fine-tuning. Article not available.

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 femtosecond 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 μm 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 hours 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.

A three-sided pyramid prism is used as a wavefront sensor in non-modulating mode with an artificial neural network (NN) as a control unit in an adaptive optical setup. The NN is composed of a convolution network for feature extraction from the pyramid signal and a three-layer feed-forward back-propagation net. The network is trained with over 15,000 examples of signal–wavefront pairs. Presenting wavefronts of 6 mm pupil diameter and a RMS error of under 200 nm to the adaptive optical system with the trained network, it can correct the aberrations within three loops to a wavefront with

A compact adaptive optics system was developed and integrated into the system for multiphoton laser scanning ophthalmoscopy. Defocus, astigmatism, trefoil, and coma are compensated via a combination of telescope and rotating pairs of phase plates. The spherical aberration as well as all dynamic aberrations are corrected by an active micro-electromechanical (MEMS) mirror. Wavefront measurement is conducted by fast SHS and pyramid sensor devices. The MEMS-mirror based adaptive optics system was validated in a study on 60 eyes of 30 subjects.The adaptive-optical multiphoton ophthalmoscope is aimed at detecting the early onset of retinal diseases such as e.g. age-related macula degeneration.

We present a method of using pairs of rotating Zernike phase plates as a precompensation unit to correct the higher-order aberrations (typically coma and trefoil) in the human eye. The rotational properties of the Zernike polynomials allow compensation of variable higher-order aberrations of the human eye with a small number of specially designed Zernike phase plates.

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 ( 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.

An analog VLSI circuit facilitating the focal point position detection in a Hartmann-Shack wavefront sensor for optical wavefront measurements is presented. Detecting the lateral deviation of each focal point of the Hartmann-Shack sensor's lens array caused by the partial distortion of the aberrated wavefront within the according lens, the phase information of the light beam can be retrieved. With the future aim of measuring optical distortions of the human eye, the ASIC is optimized to process an optical incident power of 1 nW per focal point. To prevent spatial aliasing of the focal point positions due to the eye's inherent movements during measurement, the signal processing circuits are designed to allow frame repetition rates of wavefront measurement of several hundred hertz. Experimental measurements of optical lenses show the capability of the prototype ASIC of measuring focal point position deviations relating to spherical and cylindrical wavefront aberrations with a dynamic range of /spl plusmn/1 diopters at an accuracy of /spl plusmn/0.15 diopters. The incident power of 1 nW per focal point thereby allows integration periods of 1 ms for position detection.

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.

Adaptive optics and femtosecond laser surgical systems have allowed the measurement and correction of the optical properties of the human eye, extending the knowledge of the limits of human vision.

The goal of the project “stereotactic laser-neurosurgery” is the development of a system for careful and minimal-invasive resection of brain tumors with ultrashort laser pulses through a thin probe. A confocal laser-scanning-microscope is integrated in the probe. In this paper, the simulation of its optical properties by a laboratory setup and the expansion by the ability for fluorescence microscopy are reported. For a valuation of the imaging properties, the point-spread-function in three dimensions and the axial depth-transfer-function were measured and thus, among other things, the resolving power and the capacity for depth discrimination were analysed. The microscope will enable intra-operative detection of tumor cells by the method of immunofluorescence. As a first model of the application in the brain, cell cultures, that fluorescein-labelled antibodies were bound to specifically, were used in this work. Due to the fluorescence signal, it was possible to detect and identify clearly the areas that had been marked in this manner, proving the suitability of the setup for minimal-invasive tumor diagnosis.

We investigated the use of ultrashort pulsed (femtosecond) laser technology in corneal refractive surgery. When compared to longer pulsewidth nanosecond or picosecond laser pulses, femtosecond laser-tissue interactions are characterized by significantly smaller and more deterministic photodisruptive energy thresholds, as well as reduced shock waves and smaller cavitation bubbles. We utilized a highly reliable all-solid-state femtosecond laser system for all studies to demonstrate practicality in real-world operating conditions. Contiguous tissue effects were achieved by scanning a 5-/spl mu/m focused laser spot below the corneal surface at pulse energies of approximately 2-4 /spl mu/J. A variety of scanning patterns was used to perform three prototype procedures in animal eyes; corneal flap cutting, keratomileusis, and intrastromal vision correction. Superior dissection and surface quality results were obtained for lamellar procedures (corneal flap cutting and keratornileusis). Preliminary in vivo studies of intrastromal vision correction suggest that consistent refractive changes can also be achieved with this method. We conclude that femtosecond laser technology may be able to perform a variety of corneal refractive procedures with high precision, offering advantages over current mechanical and laser devices and techniques.

A computer-guided laser probe has been developed for the application of ultrashort-pulsed lasers in neurosurgery. It is part of a novel operation concept for the treatment of deep-seated brain tumours. The system combines the positioning accuracy of stereotactic or neuronavigated instruments with the precise and therefore gentle characteristics of surgical lasers. The probe has an outer diameter of 5.5 mm and is directly inserted into the target volume. By a synchronized movement of three coaxial tubes, which guide the embedded optics, the laser radiation is focused at any time onto the current tissue surface. Since every single laser pulse has only a small effective volume of about 8 x 10(5) microm3, the application of a large number of succeeding pulses can be adapted to required geometries. Tissue fragments are removed from the growing operation cavity by continuous irrigation and suction through the laser probe. Blood vessels are detected by a confocal laser-scanning microscope, which is integrated into the probe, and can be closed by an additional coagulating laser. In this paper, the design and technical properties of the laser probe as well as its use in ablation and coagulation experiments are presented. A description of the overall operation system is given.

Nonthermal in vitro ablation of bovine neural tis- sue by using laser-induced optical breakdown generated by ultrashort laser pulses, with durations from 100 fs to 35 ps and pulse energies of up to 165mJ, has been investigated. The experiments were performed at wavelengths ranging from 630 to 1053 nm by using a femtosecond Ti:Sapphire laser, a femtosecond dye laser, and a picosecond Nd:YLF laser sys- tem. Tissue ablations have been achieved by focusing the laser beam on the surface of the tissue, to a spot diameter of 5- 20mm, resulting in the generation of a microplasma. Laser pulses from the Ti:Sapphire laser with 140 fs duration showed a two times higher efficiency of ablation than the longer 30 ps pulses from a Nd:YLF laser with an identical pulse energy. At pulse energies of 140mJ, single pass excisions deeper than 200mm were generated by the 140 fs pulses. In addition, the fluence at threshold of the ablation was found to be reduced for shorter pulse durations. For 3p slaser pulses at 630 nm, we measured the fluence at threshold to be about 5: 3J =cm 2 ; for 100 fs pulses from the same laser the experimental thresh- old was at 1: 5J =cm 2 . Histopathological examinations and scanning electron micrographs confirm the high quality of the excisions. No sign of significant thermal damage was ob- served.

Time resolved video photographs have been taken in order to investigate plasma induced surface ablation of soft tissue by infrared picosecond laser pulses with energies of about 1 mJ. The emission and propagation of shock waves in the irradiated tissue as well as in the surrounding air environment was studied. The pressure amplitudes of the shock transients were determined from the measured shock velocities. A decay of the pressure amplitude below 100 MPa was observed within a distance of about 200 \(\mu\)m from the center of laser induced optical breakdown. The dynamics of the ablation crater and the ejection of the ablation fragments was studied on a larger timescale. The maximum expansion of the ablation crater was measured to be about 200 \(\mu\)m at temporal delays of 4–5 \(\mu \)s referring to the laser pulse. Furthermore, generation and propagation of a surface deformation wave was observed. Thus, we present a detailed and consistent description of all phenomena occuring during plasma induced surface ablation of soft tissue.

The basis for most laser applications in neurosurgery is the conversion of laser light into heat when the incident laser beam is absorbed by the tissue. Irradiation of neural tissue with laser light therefore leads to its thermal damage. However, due to the diffusion of heat energy into the surrounding tissue, often there is thermal damage to neural tissue outside the area of the target volume. These are the characteristics of thermal laser/tissue interaction. In this paper we discuss how we used three different short-pulsed lasers to achieve non-thermal ablation of neural tissue. Three different short-pulsed lasers were used to generate ultrashort laser pulses in the picosecond to femtosecond range. The interaction of such laser pulses with tissue was predicted to be nonthermal. The short-pulsed lasers were used for the ablation of neural tissue using an in vitro calf brain model. The histopathological examination of the lesions revealed that the neural tissue had been removed very precisely without any sign of thermal damage to the surrounding tissue.

Threshold values of laser-induced optical breakdown (LIOB) on the surface of human corneal tissues, human enamel, and bovine brain tissues are presented. The data are obtained by using a regeneratively amplified Nd:YLF laser and a multistage dye laser system, respectively. The measured decrease in threshold fluence at shorter pulse durations is in good agreement with the authors' theoretical model.

Plasma-mediated ablations of brain tissue have been performed using picosecond laser pulses obtained from a Nd:YLF oscillator/regenerative amplifier system. The laser pulses had a pulse duration of 35 ps at a wavelength of 1.053 µm. The pulse energy varied from 90 µJ to 550 µJ at a repetition rate of 400 Hz. The energy density at the ablation threshold was measured to be 20 J/cm2. Comparisons have been made to 19 ps laser pulses at 1.68 µm and 2.92 µm from an OPG/OPA system and to microsecond pulse trains at 2.94 µm from a free running Er:YAG laser. Light microscopy and scanning electron microscopy were performed to judge the depth and the quality of the ablated cavities. No thermal damage was induced by either of the picosecond laser systems. The Er:YAG laser, on the other hand, showed 20 µm wide lateral damage zones due to the longer pulse durations and the higher pulse energies.

Der Laser stellt heute bereits eines der wichtigsten Werkzeuge in der Augenheilkunde dar. Ob man mit ihm auch Kurz-und Weitsichtigkeit ohne Komplikationen beheben kann, wird sich innerhalb der nachsten Jahre zeigen.

Die bisher entwickelten Bestrahlungstechniken von Hirntumoren sollen durch die Einfuhrung eines stereotaktischen Lasersystems zur Ablation und Absaugung eines massiven Tumors erganzt werden.

Laser scanning tomography can be used to assess retinal nerve fiber layer thickness and optic disc topography of the human eye. A pinhole is located at a plane conjugate to the focal plane of the scanning laser beam. This so-called confocal configuration assures that only the light originating from the illuminated focal plane on the retina passes through the pinhole and is detected by the photomultiplier. Consequently, images with high spatial resolution in all directions are obtained. An active optical system (active mirror) further improves the lateral/depth resolution of the laser tomographic scanner. By partially compensating for the optical aberrations introduced by the cornea and lens, the active optical system allows the illuminating beam to be enlarged to 6 mm, thus improving depth resolution twofold.

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.

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.

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.

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.

Plasma-mediated ablations were performed on human donor corneas with a short pulsed Nd:YLF laser system at 1053 nm. The pulses were 60 psec in duration at a repetition rate of 1.0 kHz. The laser beam was oriented perpendicular to the cornea surface. The threshold energy densities for ablation of epithelium, Bowman's membrane and stroma were measured. They were 6.1 +/- 1.8 J/cm2, 21.0 +/- 5.1 J/cm2 and 10.4 +/- 1.8 J/cm2, respectively. The mean rate of tissue removal at the stromal energy density threshold was about 1 micron per pulse. The walls of the laser excisions were smooth with distortions of less than 1 micron. A new quantitative model of plasma-mediated ablation is introduced and found to closely predict the observed results. Based on the promising nature of the experimental data further investigations are planned in the use of a mode locked Nd:YLF laser as an alternative to excimer lasers for refractive corneal surgery.

We describe a novel scanning laser ophthalmoscope (SLO)-based on a video-rate second harmonic generation imaging microscope. A titanium:sapphire femtosecond laser was coupled to a modified SLO. The laser beam was scanned over the sample, and the light produced by second harmonic generation (SHG) was collected for imaging at video-speed. The device was used for imaging the lamina cribrosa (LC) of enucleated pig eyes. A resolution comparable to that of commercial multiphoton microscopes was reached. The SHG images were used for determining the average pore size of the LC determined from the images; the pressure dependence of the pore size was studied by the artificial increasing of the hydrostatic pressure in the eye. A pressure increase of 44.3 mmHg enlarged the average pore size of 62 analyzed pores by a statistically significant amount. The relative pore growth was measured at four different pressure levels in 25 pores. The pressure was increased in 15 mmHg steps. A general tendency for monothonic growth was observed, although single pores grew by no means linearly.

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 µm 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. Lasers Surg. Med. 39:654–658, 2007. © 2007 Wiley-Liss, Inc.

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.

Der Laser stellt heute bereits eines der wichtigsten Werkzeuge in der Augenheilkunde dar. Ob man mit ihm auch Kurz-und Weitsichtigkeit ohne Komplikationen beheben kann, wird sich innerhalb der nachsten Jahre zeigen.

We describe existing concepts for CMOS3-based Hartmann-Shack wavefront sensors and propose the next step for a further miniaturization of adaptive optics by integrating the modal wavefront reconstruction on chip. In conventional Hartmann-Shack wavefront sensing a CCD4 camera is placed on the focal plane of a microlens array. The spot pattern in the focal plane is captured, analyzed by image processing and the wavefront is often decomposed into orthogonal modes, usually expressed in coe.cients of Zernike polynomials. The single-chip modal wavefront reconstructor would include all these processing steps in a single chip. We propose to use already existing CMOS-based wavefront sensor concepts and add a hardware arti.cial neural network for modal wavefront reconstruction.

Nd:glass femtosecond laser is promising as next generation mini-invasive eye surgical laser, with the advantages of excellent beam quality, high surgical precision and minimized side effects. However, there are still many open questions concerning the precision, efficiency and collateral effects of femtosecond laser refractive surgery. By non-invasive microscopic imaging methods including confocal, multiphoton, second harmonic and atomic force microscopy, we successfully characterized the three dimensional corneal ultrastructure without applying fixation and slicing. Based on the intrinsic properties of collagen, second harmonic cornea imaging proved to be outstanding to analyze the outcome of femtosecond laser intrastromal ablations. Strong contrast and large sensing depth second harmonic image was obtained without fixation, sectioning or labelling. The three dimensional ultrastructure of porcine cornea after Nd:glass femtosecond laser intrastromal surgery was examined to evaluate the concepts of minimum-invasive all-optical refractive eye surgery. No thermal damages were recognized and the surgical outcome appeared highly predictable. Due to the similarities between the physical principals of nonlinear laser scanning microscopy and femtosecond laser ablations, a setup of the Nd:glass femtosecond laser system integrating both the surgery and probing functions was proposed.

Wavefront technology was originally developed for the improvement of star images in optical astronomy. In 1978, this technology was for the first time applied to ophthalmology. At the University of Heidelberg, Germany, a closed loop adaptive optical system was designed to compensate for optical aberrations of the human eye. In the meantime, superresolution in retinal imaging has been achieved, providing detailed information on photoreceptors, small blood vessels, and nerve fiber structure in the human eye in vivo. Recently, new wavefront technology has been developed for the assessment of the human eye and it is now possible to apply a custom treatment to the eye, using the excimer laser. An adaptive optical closed loop system has been devised for preoperative simulation of refractive outcomes of aberration-free refractive surgical procedures. Treatment of a patient’s entire wavefront error has been demonstrated to improve the patient’s visual acuity beyond best spectacle-corrected vision.

Diode pumped Nd:glass all-solid-state femtosecond laser is promising for next generation refractive surgery, with the advantages of excellent surgical precision, minimal tissue damage and improved system stability and compactness. The microscopic evaluation of the outcome of femtosecond laser surgery is crucial before clinical applications. By two-photon laser scanning microscopy and non-invasive second harmonic imaging, the three dimensional ultrastructure of the porcine cornea is visualized without requiring slicing or staining. The minimal-invasive corneal flap cutting and non-invasive intrastromal surgery are investigated. Femtosecond laser intrastromal surgery demonstrated high ablation precision and mimimal side effects. However, there are elongated filaments/streaks observed in the cornea stroma, most likely due to the focusing optics and self-focusing.

Refractive surgery in the pursuit of perfect vision (e.g. 20/10) requires firstly an exact measurement of abberations induced by the eye and then a sophisticated surgical approach. A recent extension of wavefront measurement techniques and adaptive optics to ophthalmology has quantitatively characterized the quality of the human eye. The next milestone towards perfect vision is developing a more efficient and precise laser scalpel and evaluating minimal-invasive laser surgery strategies. Femtosecond all-solid-state MOPA lasers based on passive modelocking and chirped pulse amplification are excellent candidates for eye surgery due to their stability, ultra-high intensity and compact tabletop size. Furthermore, taking into account the peak emission in the near IR and diffraction limited focusing abilities, surgical laser systems performing precise intrastromal incisions for corneal flap resection and intrastromal corneal reshaping promise significant improvement over today's Photorefractive Keratectomy (PRK) and Laser Assisted In Situ Keratomileusis (LASIK) techniques which utilize UV excimer lasers. Through dispersion control and optimized regenerative amplification, a compact femtosecond all-solid-state laser with pulsed energy well above LIOB threshold and kHz repetition rate is constructed. After applying a pulse sequence to the eye, the modified corneal morphology is investigated by high resolution microscopy (Multi Photon/SHG Confocal Microscope).

The optical errors caused by the elements of the optical apparatus of the eye, as the cornea or the lens, strongly degrade the image on the retina. These errors are lager for larger pupil sizes.

Measurements of the wavefront of light reflected from the retina of the human eye can be used to determine optical aberrations of the human eye for large pupils. An instrument based on the Hartmann-Shack principle was developed. The wavefront is refracted by a microlens array and detected by a CCD camera. In first clinical studies human volunteer eyes and preoperative and postoperative refractive surgical patient eyes have been examined. An adaptive optical closed loop system has been devised for preoperative simulation of refractive outcomes of aberration free refractive surgical procedures. [J Refract Surg 2000; 16: S608-S6101