This PDF file contains the front matter associated with SPIE Proceedings Volume 8209, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Microglia are the resident immune cells of the central nervous system and play a crucial role in maintaining neuronal health and function. Their dynamic behavior, that is, the constant extension and retraction of microglia processes, is thought to be critical for communication between microglia and their cellular neighbors, such as neurons, astrocytes and vascular endothelial cells. Here, we investigated the morphology and dynamics of retinal microglia in vivo under normal conditions and in response to focal laser injury of blood vessel endothelial wall, using a scanning laser ophthalmoscope (SLO) designed specifically for imaging the retina of live mice. The multichannel confocal imaging system allows retinal microstructure, such as the processes of microglia and retinal vasculature, to be visualized simultaneously. In order to generate focal laser injury, a photocoagulator based on a continuous wave (cw) laser was coupled into the SLO. An acousto-optic modulator chopped pulses from the cw laser. A tip-tilt-scanner was used to direct the laser beam into a blood vessel of interest under SLO image guidance. Mild coagulation was produced using millisecond-long pulses. Microglia react dynamically to focal laser injury of blood vessel endothelial walls. Under normal conditions, microglia somas remain stationary and the processes probe a territory of their immediate environment. In response to local injury, process movement velocity approximately doubles within minutes after injury. Moreover, the previously unpolarized process movement assumes a distinct directionality towards the injury site, indicating signaling between the injured tissue and the microglia. In vivo retinal imaging is a powerful tool for understanding the dynamic behavior of retinal cells.

We present the first steps of a new measurement principle for high-precision non-contact remote and continuous monitoring of intra-ocular pressure. A photonic device involving a fast camera and a laser is presented and tested in rabbit's eyes for continuous remote monitoring of the intra-ocular pressure. The device is based on tracking the secondary speckle patterns trajectories produced by reflection of an illuminating laser beam from the iris or the sclera. The rabbit's eyes were stimulated with increasing and decreasing ramps of intra-ocular pressure. Data from the photonic device were correlated with the induced intra-ocular pressure as consequence of the infusion bag elevation variations. The measurements show a good correlation and sensitivity of the proposed device with intra-ocular pressure changes while providing a high precision measurement (5% estimated error) for the best experimental configuration.

For investigation of accommodation and presbyopia, the ciliary body and its dynamics were imaged with Optical Coherence Tomography (OCT) method. We used two OCT systems; a discretely-swept frequency-comb OCT with a center wavelength at 1.6µ and ultra-fast spectral domain OCT with a center wavelength at 1.3μm. Measurements of dynamical modification of the ciliary body were carried out with ranging in age from 22 to 79. The OCT images and movies represent the changes of the ciliary body in the relaxed state and accommodated state. Modification in the ciliary body with accommodation was more evident for the younger subject group from the results of 2D imaging. The time dependent 3D movies of ciliary body dynamics were observed for the first time using ultra-fast spectral domain OCT system. The time duration of ciliary body thickness change in relation to the accommodation was measured from the movies.

Interest in measuring peripheral refraction rapidly and accurately has been stimulated by increasing evidence that the eye's peripheral refractive state can influence axial growth. In response to this, a new clinical instrument, the EyeMapper, was developed which performs quasi real-time global (central and peripheral) refraction measurements of the human eye. The EyeMapper is an aberrometer comprising a unique deflection system to permit an extremely rapid visual field scan. Refraction measurements are taken from -50° to +50° in 10° steps within 0.45 seconds. Multiple pupil imaging paths through the deflection system provide improved lateral and axial pupil alignment, and by rotating the instrument around its main optical axis, global power maps of the eye can be generated. Using a model eye with a pivoting and translating reflective surface to simulate the peripheral and central retina, the EyeMapper was cross-validated against a conventional aberrometer (COAS-HD, Wavefront Sciences, USA) and an autorefractor (Shin-Nippon NVision K5001, Japan). In addition, the right eyes of ten participants were measured across the horizontal visual field and in one eye, refraction measurements were performed globally. Overall, the EyeMapper showed good agreement and improved repeatability when compared to the other two instruments.

Ability of a new version of one-micrometer dual-beam optical coherence angiography (OCA) based on Doppler optical coherence tomography (OCT), is demonstrated for choroidal vasculature imaging. A particular feature of this system is the adjustable time delay between two probe beams. This allows changing the measurable velocity range of moving constituents such as blood without alteration of the scanning protocol. Since choroidal vasculature is made of vessels having blood flows with different velocities, this technique provides a way of discriminating vessels according to the velocity range of their inner flow. An example of choroid imaging of a normal emmetropic eye is here given. It is shown that combining images acquired with different velocity ranges provides an enhanced vasculature representation. This method may be then useful for pathological choroid characterization.

We demonstrate an intensity-based motion sensitive method, called differential logarithmic intensity variance (DLOGIV), for 3D microvasculature imaging and foveal avascular zone (FAZ) visualization in the in vivo human retina using swept source optical coherence tomography (SS-OCT) at 1060 nm. A motion sensitive SS-OCT system was developed operating at 50,000 A-lines/s with 5.9 μm axial resolution, and used to collect 3D images over 4 mm² in a normal subject eye. Multiple B-scans were acquired at each individual slice through the retina and the variance of differences of logarithmic intensities as well as the differential phase variances (DPV) was calculated to identify regions of motion (microvasculature). En face DLOGIV image were capable of capturing the microvasculature through depth with an equal performance compared to the DPV.

None of currently used tonometers produce estimated IOP values that are free of errors. Measurement incredibility arises from indirect measurement of corneal deformation and the fact that pressure calculations are based on population averaged parameters of anterior segment. Reliable IOP values are crucial for understanding and monitoring of number of eye pathologies e.g. glaucoma. We have combined high speed swept source OCT with air-puff chamber. System provides direct measurement of deformation of cornea and anterior surface of the lens. This paper describes in details the performance of air-puff ssOCT instrument. We present different approaches of data presentation and analysis. Changes in deformation amplitude appears to be good indicator of IOP changes. However, it seems that in order to provide accurate intraocular pressure values an additional information on corneal biomechanics is necessary. We believe that such information could be extracted from data provided by air-puff ssOCT.

In this paper, we report the use of phase stabilized swept source optical coherence tomography (PhSSSOCT) for the measurement of surface mechanical wave propagation in ocular tissue in-situ. Mechanical wave propagation was measured in the mouse cornea for both young and older mice to assess tissue properties as a function of age. The measurements were performed by inducing low amplitude (< 100 μm) waves and measuring the wave attenuation at spatially distributed points using a phase-sensitive analysis of OCT signals. Obtained results indicate that the attenuation of the wave amplitude was 0.27 μm/mm in the one month-old mouse cornea and 0.37 μm/mm for 9 month old mice (and presumably of different stiffness for 1 month and 9 months old). Results also suggest that PhS-SSOCT is capable of measuring the changes in the wave amplitude as small as 0.03 μm (limited by the phase stability of the system) that allowed the measurements with a very low amplitude excitation wave, thus making the method minimally invasive. Therefore, this method could potentially be used to assess tissue biomechanical properties and to reconstruct stiffness maps of the cornea.

Confocal Brillouin spectroscopy is an innovative measurement method for the noninvasive determination of rheological tissue properties. Its application in ophthalmology can offer the possibility to determine in vivo the deformation properties of eye lens with spatial resolution. This seems to be a promising approach concerning current presbyopia research. Due to the spatially resolved detection of the viscoelastic lens properties, a better understanding of the natural aging process of the lens and the influences of different lens opacities on the stiffness is expected. Based on spectral data the refractive index profile, the protein concentration and the density profile within the lens tissue can be derived. A measurement set-up for confocal Brillouin microscopy based on spectral analysis of spontaneous Brillouin scattering signals by using a high-resolution dispersive device is presented. First in vivo measurements results on rabbit eyes are presented and evaluated concerning refractive index distribution, protein concentration, density and rheological significance. These data are compared with known research results of ex vivo lenses.

We demonstrate significantly different scattering coefficients of the retinal nerve fiber layer (RNFL) between normal and glaucoma subjects. In clinical care, SD-OCT is routinely used to assess the RNFL thickness for glaucoma management. In this way, the full OCT data set is conveniently reduced to an easy to interpret output, matching results from older (non- OCT) instruments. However, OCT provides more data, such as the signal strength itself, which is due to backscattering in the retinal layers. For quantitative analysis, this signal should be normalized to adjust for local differences in the intensity of the beam that reaches the retina. In this paper, we introduce a model that relates the OCT signal to the attenuation coefficient of the tissue. The average RNFL signal (within an A-line) was then normalized based on the observed RPE signal, resulting in normalized RNFL attenuation coefficient maps. These maps showed local defects matching those found in thickness data. The average (normalized) RNFL attenuation coefficient of a fixed band around the optic nerve head was significantly lower in glaucomatous eyes than in normal eyes (3.0mm^-1 vs. 4.9mm^-1, P<0.01, Mann-Whitney test).

Optical coherence tomography (OCT) has had a tremendous global health impact upon the current ability to diagnose, treat, and monitor multiple eye diseases. We propose that a miniature forward-imaging OCT probe can be developed for real-time ocular imaging. A miniature 25-gauge forward-imaging probe was designed and developed to use with an 850 nm spectral-domain optical coherence tomography (SDOCT) system (Bioptigen, Inc. Durham, NC). Imaging parameters were determined. Ocular tissues were examined with the miniature OCT probe. A miniature SDOCT probe was developed with the scanning driver within the hand piece. The SDOCT fiber-scanning probe maximally transmitted power of 800 μW. The scanning range was 3 mm when the probe tip was held 3 to 5 mm from the tissue surface. The axial resolution was 6 μm and the lateral resolution was 30-35 μm. The 25-gauge forward-imaging probe was used to image cellophane tape, eyelid skin, cornea, conjunctiva, sclera, iris, anterior lens, anterior chamber angle, retina, retinal tear, retinal detachment, optic nerve head, and optic nerve sheath. Images obtained from the miniature probe appeared similar to images from a 3 mm scanning range of a commercial large handheld OCT probe (Bioptigen, Inc. Durham, NC).

Recent progress in retinal image acquisition techniques, including optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO), combined with improved performance of adaptive optics (AO) instrumentation, has resulted in improvement in the quality of in vivo images of cellular structures in the outer layers of the human retina. Despite the significant progress in imaging cone and rod photoreceptor mosaics, visualization of cellular structures in the inner retina has been achieved only with extrinsic contrast agents that have not been approved for use with humans. In this paper we describe the main limiting factors in visualizing inner retinal cells and the methods we implemented to reduce their effects on images acquired with AO-OCT. These include improving the system point spread function (AO performance), monitoring of motion artifacts (retinal motion tracking), and speckle pattern reduction (temporal and spatial averaging). Results of imaging inner retinal morphology and the improvement offered by the new UC Davis AOOCT system with spatio-temporal image averaging are presented.

Optical coherence tomography with adaptive optics (AO-OCT) is a noninvasive method for imaging the living retina at the microscopic level. We used AO-OCT technology to follow changes in cone photoreceptor outer segment (OS) length and reflectance. To substantially increase sensitivity of the length measurements, a novel phase retrieval technique was demonstrated, capable of detecting changes on a nanometer scale. We acquired volume videos of 0.65°x0.65° retinal patches at 1.5° temporal to the fovea over 75 and 105 minutes in two subjects. Volumes were dewarped and registered, after which the cone intensity, OS length, and referenced phase difference were tracked over time. The reflections from inner segment/OS junction (IS/OS) and posterior tips of OS (PT) showed significant intensity variations over time. In contrast, the OS length as measured from the intensity images did not change, indicative of a highly stable OS length at least down to the level of the system's axial resolution (3μm). Smaller axial changes, however, were detected with our phase retrieval technique. Specifically, the PT-IS/OS phase difference for the same cones showed significant variation, suggesting real sub-wavelength changes in OS length of 125±46 nm/hr for the 22 cones followed. We believe these length changes are due to the normal renewal process of the cone OS that elongate the OS at a rate of about 100 nm/hr. The phase difference measurements were strongly correlated among Alines within the same cone (0.65 radians standard deviation) corresponding to a length sensitivity of 31 nm, or ~100 times smaller than the axial resolution of our system.

In ophthalmology the research using "in vitro" corneas are an excellent model for studies of new ophthalmologic procedures, enabling the analysis of effectiveness, performance and even safety parameters of the procedure. In this work we studied four "in vitro" human corneas preserved in OPTISOL-GS, with initial average pachymetry of 542 microns and a post-mortem average of 6 days. The corneas were preserved in OPTISOL-GS and were washed with saline solution to remove the excess the preservative medium. The corneas were placed in a device aligned with an ultraviolet source of 3mw/cm2 and an optical fiber positioned after the device near the endothelium of the cornea. The UV transmittance spectra in the region of 360-370nm were captured by the emission of UV source for 3 seconds. These spectra were captured every 5 minutes in a total of 60 minutes, resulting in 13 spectra per cornea. The measured average initial UV transmittance was 73% and after 50 minutes of dehydration there was no significant difference in the corneal teansmittance properties. However, for the last 10 minutes we have observed a decrease in the UV transmittance of 4%, probably indicated by corneal dehydration and swelling (wrinkling of the cornea tissue. The final average pachymetry was 421 microns and the UV transmittance after the 60 minutes was 69%. Therefore we can suppose that the UV transmittance of corneas "in vitro" is invariant over a period of up to 60 minutes, even with the thickness decrease, since the material that absorbs in the UV region remains intact and only water loss occurs.

The measurements of the transmittance of ultraviolet and infrared radiation through sunglasses are standard requirements for certification of these lenses. According to Brazilian Standard NBR15111(2004), the electromagnetic spectrum relative to UVA, UVB and UVC (100 - 400nm) and infrared (700-1400nm) must be protected, by filters, according to the lens category. The categories are in a scale of 0 to 4, according to the amount of visible light transmitted through the sunglasses. An opto-electronic set up was assembled in this work, using light sources (set of LEDs), which cover the electromagnetic spectrum in the range of 380nm - 780nm; one visible light sensor for measuring the visible light transmission through the lens of the sunglasses; and an electronic circuit to control the intensity of the LEDs light. The device performs the calibration of the light source to match the requirements by the standard. The prototype has an accuracy of 0.1% for transmission; resolution of 0.1% and correlation factor of r² = 0.991 for the tested lenses compared to CARY 5000 - Varian spectrophotometer.

The transmittance of UVA light through the human preserved cornea of over 400μm thickness during the corneal collagen cross-linking procedure has been measured spectroscopically. The 25 corneas, (average thickness of 570 μm), preserved in OptisolGS, were washed with saline, desepithelization was performed, and the cornea was laid on the lid of a Chiron Ophthalmics corneal storage chamber. A UV-VIS optical fiber was positioned at the crystalline position (10mm after the endothelium) and fixed in a 3mm hole of the chamber and then connected to a spectrophotometer to detect the amount of delivered UVA light on the endothelium. Current procedure protocol was performed, i.e., one drop of riboflavin 0.1%, 400 mOsm, was applied on the naked cornea, every 5 minutes (total of 12 drops). The UV irradiation (365±5 nm, 3mW/cm2, 1.51 mW, 5.405 J/cm2) was performed after 30 min of instillation for an additional 30 min. The average transmittance of the desepithelized cornea without Riboflavin at the crystalline position is 65.8%'; after the 1st drop of Riboflavin, transmittance is 51.4%; after 2nd drop, 46.1%; after 3rd drop, 41.9% ; after 4th drop, 38.7%; after 5th drop, 35.9%; after 6th drop 33.6% ; after 7th drop, 31.0%; after 8th drop; 28.8%; after 9th drop, 27.2%; after 10h drop, 25.4%; after 11th drop, 23.9%; and finally after 12th drop, 22.5%. The average transmittance in terms of energy during the 30 min irradiation procedure fluctuated from 0.930 to 0.675mW/cm2.

The measurement of blood flow in the ocular fundus is of scientific and clinical interest. Investigating ocular blood flow in the choroid may be important to understand the pathogenesis of numerous ocular diseases, such as glaucoma or agerelated macular degeneration (AMD). Laser Doppler flowmetry (LDF) was applied to measure relative velocity, volume and flux of red blood cells in the tissues of human eye. Its main application lies in the possibility of assessing alterations in blood flow early in the course of diseases. The purpose of the present study was to investigate the effect of pupil dilatation with one drop of 1% tropicamide on blood flow in the foveal region of the choroid of the human fundus. The blood flow parameters were measured in 24 eyes during 30 minutes (one measurement in every 3 minutes) after the application of the drop. Since the Doppler parameters depend on the scattering geometry, which may change as the pupil dilates; an artificial pupil of 4mm in diameter was placed directly in front the eye. Following the administration of tropicamide the mean pupil diameter was increased from 3.29 mm to 8.25 mm (P<0.0001, Paired student t-test). In comparison to the baseline values, the data shows no significant increases were observed in velocity, volume, and flow with 4 mm artificial pupil (0.2%, 1.3%, 0.8% respectively) and a statistically significant increases were observed without artificial pupil (10.7%, 13.9%, 12.8% respectively) following the application of tropicamide.

An ophthalmic instrument for innovative analyses of the eye microcirculation is presented. The developed system employs a laser source to performs the DWS (diffusing-wave-spectroscopy) analysis of the light intensity back diffused by the ocular fundus structures, thus allowing a noninvasive detection of changes and modulations of both optical and dynamical properties of blood flow. The reported preliminary experimental results recorded in-vivo on rabbits' ocular fundus, demonstrate the ability of the system to detect nano- micro-aggregates carried by the blood flow in the ocular fundus.

Femtosecond laser sculpturing of corneal tissue is commonly used for the preparation of endothelial flaps. Diode laser welding of ocular tissues is a procedure that enables minimally invasive suturing of tissues. The combination of these laser based techniques results in a new approach to minimally invasive ophthalmic surgery, such as in endothelial corneal transplant (or endothelial keratoplasty - EK). In this work we present the "all laser" EK performed in human subjects. 24 pseudophakic patients with bullous keratopathy underwent EK: the femtosecond laser was used to prepare the 100 ìm thick and 8.5 mm diameter donor Descemet endothelial flap. After staining the stromal layer of the donor flap with a liquid ICG solution, the donor flap was inserted in the recipient eye by the use of the Busin injector. Then, the endothelial layer was laser-welded to the recipient eye (10 laser spots around the periphery of the flap), in order to reduce the risk of postoperative dislocation of the transplanted flap. A transplanted flap engraftment was observed in all the treated eyes. The staining procedure used to perform laser welding also enabled to evidence the stromal side of the donor flap, so as the flap was always placed in the right side position. The endothelial cells counts in both the laserwelded flaps and in a control group were in good agreement. The proposed technique is easy to perform and enables the reduction of postoperative endothelial flap dislocations.

We report the evaluation of water film on a contact lens using an improved optical reflectometry technique. A galvanometer scanner is added to an optical reflectometry system for fast measurement beam alignment. Light from a Tungsten Halogen light source travel down a 2×1 fiber coupler, go through the focusing lens and the galvanometer scanner, and light up the water film on the contact lens. The air/water and water/contact lens interfaces reflect the light back to the fiber, where the spectral dependent reflection data is acquired by the fiber coupled spectrometer for analysis. From the reflective spectra, the water film thickness can be calculated using predictor-corrector curve fitting method. In the scanning selection and the curve fitting calculation, a band stop filter is applied to the reflectance spectrum to eliminate data noise.

The development of affordable means to image the retina is an important step toward the implementation of eye disease screening programs. In this paper we present the i-RxCam, a low-cost, hand-held, retinal camera for widespread applications such as tele-retinal screening for eye diseases like diabetic retinopathy (DR), glaucoma, and age-related ocular diseases. Existing portable retinal imagers do not meet the requirements of a low-cost camera with sufficient technical capabilities (field of view, image quality, portability, battery power, and ease-of-use) to be distributed widely to low volume clinics, such as the offices of single primary care physicians serving rural communities. The i-RxCam uses a Nikon D3100 digital camera body. The camera has a CMOS sensor with 14.8 million pixels. We use a 50mm focal lens that gives a retinal field of view of 45 degrees. The internal autofocus can compensate for about 2D (diopters) of focusing error. The light source is an LED produced by Philips with a linear emitting area that is transformed using a light pipe to the optimal shape at the eye pupil, an annulus. To eliminate corneal reflex we use a polarization technique in which the light passes through a nano-wire polarizer plate. This is a novel type of polarizer featuring high polarization separation (contrast ratio of more than 1000) and very large acceptance angle (>45 degrees). The i-RxCam approach will yield a significantly more economical retinal imaging device that would allow mass screening of the at-risk population.

Commercial OCT systems provide pachymetry measurements. Full corneal topographic information of anterior and posterior corneal surfaces for use in cataract surgery and refractive procedures is a desirable goal and would add to the usefulness of anterior and posterior segment evaluation. While substantial progress has been made towards obtaining "average" central corneal power (D Huang), power in different meridians and topography are still missing. This is usually reported to be due to eye movement. We analyze the role of centration, eye movements and develop a model that allows for the formulation of criteria for obtaining reliable topographic data within ¼ diopter.

A laser Doppler module easily integrated into a commercial ophthalmic microscope is proposed. Such setup adds flow measurement capability to standard visual inspection of the fundus. The proposed instrument may provide important clinical information such as the detection of vessel occlusion provided by surgical treatments (i.e. photocoagulation). The measuring system is based on a self-mixing laser diode Doppler flowmeter (SM-DF). Reduced costs, easy implementation and small size represent the main features of SM-DF. Moreover, this technique offers the advantage to have the excitation and measurement beams spatially overlapped, thus both overcoming the alignment difficulty of traditional laser Doppler flowmeter and, well fitting with to limited optical aperture of the pupil. Thanks to an on-board DSP-microcontroller, the optoelectronic module directly estimates the blood flow; USB connection and an ad-hoc developed user-friendly software interface allow displaying the result on a personal computer. Preliminary test demonstrates the applicability of the proposed measuring system.

High absorption property of tissues in the IR range (λ> 2 μm) results in effective tissue ablation, especially near 3 μm. In the mid-infrared range, wavelengths of 6.1 μm and 6.45 μm fall into the absorption bands of the amide protein groups Amide-I and Amide-II, respectively. They also coincide with the deformation mode of water, which has an absorption peak at 6.1 μm. This coincidence makes 6.1 μm laser a better ablation tool that has promising effectiveness and minimum collateral damages than 3 μm lasers. In this work, we performed bovine corneal ablation test in-vitro using high-power 6.1μm quantum cascade laser (QCL) operated at pulse mode. Quantum cascade laser has the advantages of low cost, compact size and tunable wavelength, which makes it great alternative Mid-IR light source to conventional tunable free-electron lasers (FEL) for medical applications. Preliminary results show that effective corneal stroma craters were achieved with much less collateral damage in corneal tissue that contains less water. Future study will focus on optimizing the control parameters of QCL to attain neat and precise ablation of corneal tissue and development of high peak power QCL.

The design of robust Adaptive Optics systems (AO) requires to characterize spatially and temporally the aberrations. Thus, it is of importance to have an instrument able to measure the aberrations at high spatial and temporal resolutions. The high spatial resolution is necessary to have an extended modal decomposition of aberrations, to characterize finely the pupil irradiance and to quantify aliasing and fitting errors. The high temporal resolution is necessary to analyze the evolution of very fast phenomena contributing in the aberration dynamics, e.g. fast pupil movements, tear film and accommodation. Because, Hardware constraints make it difficult to obtain high spatial and temporal resolutions on a single detector, we have designed a new aberrometer comprising two synchronized instruments, one highly spatially resolved, the second one highly temporally resolved that allows to perform such measurements. Preliminary results have been obtained on the highly spatially resolved instrument. The integration of the second instrument is in progress. An overview of the first instrument results is presented in this paper.