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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.

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.

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.

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.

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.

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.

Time-resolved video photographs have been taken in order to investigate plasma induced surface ablation of soft tissue by IR 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 micrometers 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 time scale. The maximum expansion of the ablation crater was measured to be about 200 (Mu) m at temporal delays of 4-5 microsecond(s) after the impact of 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 occurring during plasma-mediated surface ablation of soft tissue.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Three nonlinear (beta) -barium borate (BBO) crystals were used to generate the fourth and fifth harmonic of a Nd:YLF picosecond laser with a fundamental wavelength of 1053 nm. The UV laser pulses were used to study the surface ablation of bovine soft tissue. Whereas the general study was concentrated on brain tissue, first results were also obtained with cornea tissue. The ablation efficiency as a function of pulse energy and the ablation threshold were determined for both UV wavelengths. Histology and SEM- microscopy of the performed laser lesions are presented. Furthermore, time-resolved flash photographs we recorded by illuminating the ablation area with green picosecond laser pulses and detecting the probe beam with a CCD chip. The photographs yielded information about the physical properties of the ablation process. The emission of a shock wave into the air environment as well as the ejection of dissected ablation fragments was observed for pulse energies down to 10 (mu) J. Both UV wavelengths can be combined with an ophthalmic application system, which comprises an xyz- scanning unit for realizing precalculated ablation patterns and a z-tracker for detecting the current surface position of the cornea. The information of the z-tracker can be used to compensate the axial eye-movement with the xyz-scanning unit.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

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.

A microprocessor controlled photo-thermoplastic device has been developed for the real-time recording of holographic filters in a hybrid digital-optical data processing system. It is possible to position the filters with an accuracy of 0,2 µm.© (1981) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

To study the retinal surface in the human eye in normal and diseased states we used laser scanning tomography. The confocal arrangement of the laser tomographic scanner permits examination of retinal topography in the axis perpendicular to the retinal surface. The eyes examined with the laser tomographic scanner included normal eyes, eyes with macular holes, impending macular holes, radiation retinopathy, macular edema, photocoagulation scars, subfoveal scars, and serous detachment of the fovea associated with subretinal neovascularization. The laser tomographic scanner is a new method that allows measurements of the topography of the internal limiting membrane in the macular area and may improve our understanding of the pathophysiologic characteristics and treatment of a variety of disorders of the macula.