Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss.

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

In-vivo retinal imaging with off-axis full-field time-domain optical coherence tomography.

With a simple setup, mainly composed of a low coherence light source and a camera, full-field optical coherence tomography (FF-OCT) allows volumetric tissue imaging. However, fringe washout constrains its use in retinal imaging. Here, we present a novel motion-insensitive approach to FF-OCT, which introduces path-length differences between the reference and the sample light in neighboring pixels using an off-axis reference beam. The temporal carrier frequency in scanned time-domain OCT is replaced by a spatial carrier frequency. Volumetric in-vivo FF-OCT measurements of the human retina were acquired in only 1.3 s, comparable to the acquisition times of current clinically used OCT devices.

Two-photon microscopy using fiber-based nanosecond excitation.

Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.

Twisted-nematic liquid-crystal-on-silicon adaptive optics aberrometer and wavefront corrector.

A Hartmann-Shack wavefront sensor (HSWS) has been proven to be a reliable tool for the quantitative analysis of human ocular aberrations. In an active adaptive optics (AO) system, it has the role to monitor wave aberrations. To ensure the exclusive retrieval of Zernike coefficients for the measured ocular wavefronts, we first nullify the AO system's aberrations. This is of particular importance in our setup with a twisted-nematic (TN) liquid-crystal-on-silicon (LCoS) chip as the wavefront manipulator due to its strong unwanted zero-order diffractive beam. We characterize the AO system's performance-before and after ocular corrections-by means of different parameters, including experimental and simulated point spread functions (PSFs). An iterative closed-loop algorithm reduces the residual wavefront error to typical values of 0.1 mum. This system constitutes a wavefront corrector that can possibly be used for high resolution retinal imaging purposes or for visual psychophysical experiments.

In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells.

The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

General purpose control system for scanning laser ophthalmoscopes.

A flexible control system for scanning laser ophthalmoscopes is described that is quick and simple to configure, easily modified or adapted, and containing many useful features. The system facilitates adjustment of several parameters to account for changes to the scan position, ambient light and temperature, including both optical and electronic components, which is otherwise difficult and time-consuming to perform. The system is portable and uses custom-designed printed circuit boards. All system parameters, such as focus, scan rate,scan depth and stereo control can be digitally controlled from a computer via a single serial port. Custom software allows changes to any system parameters by simply sending the required control data to the rack. The circuit boards in the system are multilayer,incorporating good ground-plane techniques to minimize noise, programmable logic and semicustom logic for low cost and compact size, and microcontrollers with embedded firm ware for flexible operation. Retinal images demonstrate that the system performs well.