A recent study showed that 355-nm nanosecond lasers cut cornea with similar precision to infrared femtosecond lasers. However, use of ultraviolet wavelength requires precise assessment of ocular safety to determine the range of possible ophthalmic applications. In this study, the 355-nm nanosecond laser was evaluated for corneal and iris damage in rabbit, porcine, and human donor eyes as determined by minimum visible lesion (MVL) observation, live/dead staining of the endothelium, and apoptosis assay. Single-pulse damage to the iris was evaluated on porcine eyes using live/dead staining. In live rabbits, the cumulative median effective dose (ED50) for corneal damage was 231 J / cm2, as seen by lesion observation. Appearance of endothelial damage in live/dead staining or apoptosis occurred at higher radiant exposure of 287 J / cm2. On enucleated rabbit and porcine corneas, ED50 was 87 and 52 J / cm2, respectively, by MVL, and 241 and 160 J / cm2 for endothelial damage. In human eyes, ED50 for MVL was 110 J / cm2 and endothelial damage at 453 J / cm2. Single-pulse iris damage occurred at ED50 of 208 mJ / cm2. These values determine the energy permitted for surgical patterns and can guide development of ophthalmic laser systems. Lower damage threshold in corneas of enucleated eyes versus live rabbits is noted for future safety evaluation.
Electronic retinal prostheses represent a potentially effective approach for restoring some degree of sight in blind
patients with retinal degeneration. Functional restoration of sight would require hundreds to thousands of electrodes
effectively stimulating remaining neurons in the retina. We present a design of an optoelectronic retinal prosthetic
system having 3mm diameter retinal implant with pixel sizes down to 25 micrometers, which allows for natural eye
scanning for observing a large field of view, as well as spatial and temporal processing of the visual scene to optimize
the patient experience. Information from a head mounted video camera is processed in a portable computer and
delivered to the implanted photodiode array by projection from the LCD goggles using pulsed IR (810 nm) light. Each
photodiode converts pulsed light (0.5 ms in duration) into electric current with efficiency of 0.3 A/W using common bi-phasic
power line. Power is provided by the inductively-coupled RF link from the coil on the goggles into a miniature
power supply implanted between the sclera and the conjuctiva, and connected to subretinal implant with a thin 2-wire
3-dimensional structures in the subretinal prosthesis induce retinal migration and thus ensure close proximity between
stimulating electrodes and the target retinal neurons. Subretinal implantations of the 3-dimentional pillar and chamber
arrays in RCS rats with 2 and 6 week follow-up demonstrate achievement of intimate proximity between the stimulation
cites and the inner nuclear layer. In some instances formation of a fibrotic seal has been observed.
Electronic retinal prostheses represent a potentially effective approach for restoring some degree of
sight in blind patients with retinal degeneration. However, levels of safe electrical stimulation and the
underlying mechanisms of cellular damage are largely unknown. We measured the threshold of cellular
damage as a function of pulse duration, electrode size, and number of pulses to determine the safe range of
stimulation. Measurements were performed in-vitro on embryonic chicken retina with saline-filled glass
pipettes for stimulation electrodes. Cellular damage was detected using Propidium Iodide fluorescent
staining. Electrode size varied from 115μm to 1mm, pulse duration from 6μs to 6ms, and number of pulses
from 1 to 7,500. The threshold current density was independent of electrode sizes exceeding 400μm. With
smaller electrodes the current density was scaling reciprocal to the square of the pipette diameter, i.e. acting
as a point source so that the damage threshold was determined by the total current in this regime. The
damage threshold current measured with large electrodes (1mm) scaled with pulse duration as t<sup>-0.5</sup>, which is
characteristic of electroporation. For repeated electrical pulsed exposure on the retina the threshold current
density varied between 0.059 A/cm2 at 6ms to 1.3 A/cm2 at 6μs. The dynamic range of safe stimulation,
i.e. the ratio of damage threshold to stimulation threshold was found to be duration-dependent, and varied
from 10 to 100 at pulse durations varying between 10μs to 10ms. Maximal dynamic range of 100 was
observed near 1ms pulse durations.
A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery
techniques. Viral delivery has encountered a number of serious problems including immunological reactions and
malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo
or produce severe collateral damage to ocular tissues.
We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation.
For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient
transfection of RPE without visible damage to the retina.
Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor
10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous
potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and
efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.
Development of the electronic retinal prosthesis for restoration of sight in patients suffering from the degenerative retinal diseases faces many technological challenges. To achieve significant improvement in the low vision patients the visual acuity of 20/80 would be desirable, which corresponds to the pixel size of 20μm in the retinal implant. Stimulating current strongly (quadratically) depends on distance between electrode and cell. To achieve uniformity in stimulation thresholds, to avoid erosion of the electrodes and overheating of tissue, and to reduce the cross-talk between the neighboring pixels the neural cells should not be separated from electrodes by more than a few micrometers. Achieving such a close proximity along the whole surface of an implant is one of the major obstacles for the high resolution retinal implant.
To ensure proximity of cells and electrodes we have developed a technique that prompts migration of retinal cells towards stimulating sites. The device consists of a multilayered membrane with an array of perforations of several (5-15) micrometers in diameter in which addressable electrodes can be embedded. In experiments in-vitro using explants of the whole retina of P7 rats, and in-vivo using adult rabbits and RCS rats the retinal tissue grew into the pores when membranes were positioned on the sub-retinal side. Histology has demonstrated that migrating cells preserve synaptic connections with cells outside the pores, thus allowing for signal transduction into the retina above the implant.
Intimate proximity of cells to electrodes achieved with this technique allows for reduction of the stimulation current to 2μA at the 10μm electrode. A 3mm disk array with 18,000 pixels can stimulate cells with 0.5 ms pulses at 50Hz while maintaining temperature rise at the implant surface below 0.3°C. Such an implant can, in principle, provide spatial resolution geometrically corresponding to the visual acuity of 20/80 in a visual field of 10°.
Capturing, separation and removal of thin, evasive, and often transparent membranes attached to the underlying tissue is typically a very difficult task in vitreoretinal surgery. The most challenging part of such procedures is in initial separation of the membrane, which then allows for a strong grip of the micro-tweezers holding it from two sides. Attempts of performing this procedure often lead to piercing and otherwise damaging the underlying tissue. Accordingly, there is a need for devices that could attach to tissue in a minimally-traumatic manner approaching it from only one side. It is desirable that such a device would attach to a tissue on a push of a button and release it on demand.
We developed a technique that allows for strong attachment of an electrode to tissue with a single electrical pulse, and disconnection of it from the tissue with a different pulse. Adhesion does not require any electrical support after the pulse, and the adhesive forces generated on a wire electrode of 50 micrometer in diameter are sufficient for manipulation of all types of cellular and non-cellular intraocular tissues. To reduce electroporation-related tissue damage the bipolar train of pulses is applied with burst duration 50-200 microsecond. At optimal pulse parameters the tissue damage is limited to a single layer of cells adjacent to the surface of electrode.
Electrically-induced adhesion is very convenient for lifting and manipulation of vitreoretinal membranes. It can also be used for attachment of a needle to a membrane and injection of liquid into the sub-membrane space, thus separating the membrane from the underlying tissue without peeling. Similarly, injection of medication into small retinal blood vessels can be performed without insertion of the needle inside the blood vessels.
Introduction: Light Scattering Spectroscopy has been a recently developed as a non-invasive technique capable of sizing the cellular organelles. With this technique, we monitor the heat-induced sub-cellular structural transformations in a human RPE cell culture.
Material and Methods: A single layer of human RPE cells (ATCC) was grown on a glass slide. Cells are illuminated with light from a fiber-coupled broadband tungsten lamp. The backscattered (180 degree) light spectra are measured with an optical multichannel analyzer (OMA). Spectra are measured during heating of the sample.
Results: We reconstructed the size distribution of sub-micron organelles in the RPE cells and observed temperature-related changes in the scattering density of the organelles in the 200-300nm range (which might be peroxisomes, microsomes or lysosomes). The sizes of the organelles did not vary with temperature, so the change in scattering is most probably due to the change in the refractive indexes. As opposed to strong spectral variation with temperature, the total intensity of the backscattered light did not significantly change in the temperature range of 32-49 °C.
Conclusion: We demonstrate that Light Scattering Spectroscopy is a powerful tool for monitoring the temperature-induced sub-cellular transformations. This technique providing an insight into the temperature-induced cellular processes and can play an important role in quantitative assessment of the laser-induced thermal effects during retinal laser treatments, such as Transpupillary Thermal Therapy (TTT), photocoagulation, and Photodynamic Therapy (PDT).
Precise and tractionless tools are needed for cutting and ablation of ocular tissue in such operations as vitreoretinal surgery, capsulotomy, non-penetrating trabeculectomy and many others. Previously we reported about the Pulsed Electron Avalanche Knife capable of tractionless dissection of soft tissue in liquid media using the 100 ns-long plasma-mediated electric discharges applied via a 25 um inlaid disk electrode. In this work we present a next step in the development of this technique, which dramatically improves its precision, the cutting rate and the scope of applicability.
(1) Due to spherical geometry of the discharge with the disk-like microelectrode the width of the cut was equal to its depth. To overcome this limitation we apply now a thin cylindrical electrode where the width and the depth of the cut are controlled independently. (2) Cavitation accompanying the sub-microsecond explosive evaporation was a major limiting factor in precision of this technique. In a new modality we apply bursts of pulses, which allow for much higher energy deposition without increase in the size of the transient vapor cavity. (3) Coagulation regime for blood vessels larger than 25 microns in diameter was not possible in the initial approach. It is now available due to extension of the electrode in one dimension. (4) Increase in pulse duration up to several tens of microseconds allows for reduction in voltage and, consequently, in width of the insulator. This, in turn, enables development of the ultra-thin electrodes that can be applied via an intraocular endoscope or 25 G needles. The new device was found capable of rapidly and precisely dissecting virtually all types of ocular tissue: from soft membranes to cornea and sclera. In addition to vitreoretinal surgery it applications can now expand into anterior chamber surgery including capsulotomy and trabeculectomy.
Cavitation bubbles accompany explosive evaporation of water after pulsed energy deposition during endosurgery. Bubbles collapsing at the time of an endo-probe produce a powerful and damaging water jet propagating forward in the axial direction of the probe. We demonstrate that formation of this flow and associated tissue damage can be prevented by application of the concave probes that slow the propagation of the back boundary of the bubble. A similar effect can be achieved by positioning an obstacle to the flow, such as a ring or a pick tip in a close proximity to the back, side or front of the tip. Dependence of the flow dynamics on geometry of the probe was studied using fast flash photography and particle velocimetry. With a flat tip a maximal jet velocity of 80 m/s is achieved at a pulse energy of 0.12 mJ, while with an optimized concave probe the jet is completely stopped. The maximal distance between the probe and the tissue at which cells were affected by the water jet was measured using choriallantoic membrane of a chick embryo and Propidium Iodide staining. Changing the tip geometry from flat or convex to an optimized concave shape resulted in reduction of the damage distance by a factor of 4 with pulse energies varying from 0.02 to 0.75 mJ. Elimination of the water jet dramatically improves precision and safety of the pulsed endosurgery reducing the axial damage zone to a size of the cavitation bubble at its maximal expansion.
The use of a new method of interferometry of chirped pulse for precise remote measurement of lengths and velocities is proposed, evaluated and tested in experiments. The potentialities of the method in comparison with conventional methods are considered, and its possible applications are discussed.
The use of self phase modulation for spectrum broadening with subsequent pulse compression by diffraction gratings allow to compress high energy laser pulses in wide range of pulse lengths. We have discussed different feasibility of such method for pulse shortening.
A high-power all-Nd:glass laser using fiberless chirped-pulse- amplification technique is presented. The laser is capable of producing 1.2-ps, 2-TW pulses while the effective divergence of the beam does not exceed 10<SUP>-4</SUP> rad.
While the optical breakdown of transparent dielectrics for nanosecond and longer laser pulses is well investigated theoretically and experimentally, the breakdown in a field of picosecond and femtosecond pulses has not been adequately explored. At the same time such investigation presents scientific interest, because short pulse duration can make some change for a mechanism of the optical breakdown. Also these data have practical importance for high energy laser development and management of temporal parameters of a pulse. Thus we have carried out measurements of the damage thresholds for pulse durations 450 ps and 3 ps (FWHM) for fused silica. Theoretical analysis is presented to explain experimental results. The possibility of applying the optical breakdown for pulse shortening is discussed.
In the present work the interaction of single and series of laser pluses with laser energy to 0.5 J (for different light polarizations and incident angles) with aluminum target was investigated. It was found out, that p-polarized laser light is absorbed much better than s-polarized light. It is a result of resonance absorption. The main part of absorbed laser energy is spent on heating and fast particle generation. X-ray intensity is higher in the case of p-polarized laser light than in the case of s-polarized laser light. Results of numerical calculations by codes 'SKIN' and 'ION' are in good agreement with the experimental ones.
The construction of 1 TW Nd:glass laser system with pulse compression is described. The laser system consists of master oscillator with self-mode locking and negative feed-back, stretcher, regenerative amplifier, two preamplifiers, amplifier chain and diffraction gratings compressor. The possibilities of improving the laser system parameters are discussed.