This dissertation documents the development of a new illumination technique for use in the studies of retinal oximetry and fundus spectroscopy. Intravitreal illumination is a technique where the back of the eye is illuminated trans-sclerally using a scanning monochromator coupled into a fiber optic illuminator. Retinal oximetry is the processof measuring the oxygen saturation of blood contained in retinal vessels by quantitative measurement of the characteristic color shift seen as blood oxygen saturation changes from oxygenated blood (reddish) to deoxygenated blood (bluish). Retinal oximetry was first attempted in 1963 but due to a variety of problems with accuracy and difficulty of measurement, has not matured to the point of clinical acceptabilityor commercial viability.Accurate retinal oximetry relies in part on an adequate understanding of the spectral reflectance characteristics of the fundus. The use of intravitreal illumination allows new investigations into the spectral reflectance properties of the fundus. The results of much research in fundus reflectance and retinal oximetry is detailed in thisdocument, providing new insight into both of these related fields of study.Intravitreal illumination has been used to study retinal vessel oximetry and fundus reflectometry resulting in several important findings that are presented in this document. Studies on enucleated swine eyes have provided new insight into the bidirectional reflectance distribution function of the fundus. Research on live swine hasshown accurate measurement of retinal vessel oxygen saturation and provided the first in vivo spectral transmittance measurement of the sensory retina. A secondary discovery during this research suggests that vitrectomy alters the retinal vasculature,a finding that should spawn new research in its own right.

No / The purpose of the research was to examine the practical feasibility of developing a hyperspectral camera from a Zeiss fundus camera and to illustrate its use in imaging diabetic retinopathy and glaucoma patients. The original light source of the camera was replaced with an external lamp filtered by a fast tunable liquid-crystal filter. The filtered light was then brought into the camera through an optical fiber. The original film camera was replaced by a digital camera. Images were obtained in normals and patients (primary open angle glaucoma, diabetic retinopathy) recruited at the Manchester Royal Eye Hospital. A series of eight images were captured across 495- to 720-nm wavelengths, and recording time was less than 1.6s. The light level at the cornea was below the ANSI limits, and patients judged the measurement to be very comfortable. Images were of high quality and were used to generate a pixel-to-pixel oxygenation map of the optic nerve head. Frame alignment is necessary for frame-to-frame comparison but can be achieved through simple methods. We have developed a hyperspectral camera with high spatial and spectral resolution across the whole visible spectrum that can be adapted from a standard fundus camera. The hyperspectral technique allows wavelength-specific visualization of retinal lesions that may be subvisible using a white light source camera. This hyperspectral technique may facilitate localization of retinal and disc pathology and consequently facilitate the diagnosis and management of retinal disease.

V první části se práce se zabývá rešerší metod a principů potřebných při registraci obrazu. Dále pak popisuje zobrazovací systémy očního pozadí jako jsou OCT, fundus kamera a SLO. Druhá část práce je zaměřena na praktickou realizaci programů pro registraci snímků z OCT, SLO a fundus kamery v programovém prostředí Matlab.

Este trabalho apresenta as metodologias que foram empregadas no desenvolvimento de um equipamento de retinografia digital. O sistema proposto foi inteiramente desenvolvido, e manufaturado no Brasil, tornando-se o primeiro retinógrafo digital com tecnologia nacional. O equipamento descrito nesta dissertação é capaz de realizar tanto retinografias, que são imagens capturadas para observação do fundo de olho, quanto angiografias, que são exames nos quais, por meio da administração de contrastes, é possível destacar estruturas específicas da retina. Estes procedimentos permitem o diagnóstico de diversas patologias relacionadas não só à retina, mas também a diversas outras estruturas do olho humano, como por exemplo, a coróide. Esta dissertação demonstra alguns dos desafios e métodos empregados no desenvolvimento do equipamento proposto, além de uma visão geral de cada um dos sistemas que o compõem. São descritos os sistemas mecânico, óptico, eletrônico e de software, sendo cada um deles detalhados quanto às principais funções que desempenham no equipamento. Também são descritas as ferramentas de desenvolvimento utilizadas durante as etapas de análise e projeto. Ainda, são apresentados os testes de desempenho do equipamento, bem como um comparativo entre o sistema proposto e alguns dos equipamentos que atualmente são considerados referência entre médicos e operadores. / This work presents the methodology applied to the development of a digital fundus camera. The proposed system was entirely developed and manufactured in Brazil, becoming the first digital fundus camera with national technology. The described equipment can perform fundus images, which are images captured from eye fundus observation, and also angiograms, which are exams performed through the admission of dyes, where particular retinal structures are enhanced. Those procedures allow the diagnosis of several pathologies not only related to the eye fundus, but also many different structures of the human eye, like the choroid. This dissertation shows some of the challenges and methods that were applied to the development of the proposed system, in addition to an overview of each of the systems that composes the equipment. The mechanical, optical, electronic and software systems are described, being each one of them detailed regarding its main features performed in the equipment. Development tools used during the design are also described. Still, the functional and performance tests and results are described, and also a comparative evaluation between the proposed system and some of the equipments currently defined as reference among physicians and users.

Este trabalho apresenta as metodologias que foram empregadas no desenvolvimento de um equipamento de retinografia digital. O sistema proposto foi inteiramente desenvolvido, e manufaturado no Brasil, tornando-se o primeiro retinógrafo digital com tecnologia nacional. O equipamento descrito nesta dissertação é capaz de realizar tanto retinografias, que são imagens capturadas para observação do fundo de olho, quanto angiografias, que são exames nos quais, por meio da administração de contrastes, é possível destacar estruturas específicas da retina. Estes procedimentos permitem o diagnóstico de diversas patologias relacionadas não só à retina, mas também a diversas outras estruturas do olho humano, como por exemplo, a coróide. Esta dissertação demonstra alguns dos desafios e métodos empregados no desenvolvimento do equipamento proposto, além de uma visão geral de cada um dos sistemas que o compõem. São descritos os sistemas mecânico, óptico, eletrônico e de software, sendo cada um deles detalhados quanto às principais funções que desempenham no equipamento. Também são descritas as ferramentas de desenvolvimento utilizadas durante as etapas de análise e projeto. Ainda, são apresentados os testes de desempenho do equipamento, bem como um comparativo entre o sistema proposto e alguns dos equipamentos que atualmente são considerados referência entre médicos e operadores. / This work presents the methodology applied to the development of a digital fundus camera. The proposed system was entirely developed and manufactured in Brazil, becoming the first digital fundus camera with national technology. The described equipment can perform fundus images, which are images captured from eye fundus observation, and also angiograms, which are exams performed through the admission of dyes, where particular retinal structures are enhanced. Those procedures allow the diagnosis of several pathologies not only related to the eye fundus, but also many different structures of the human eye, like the choroid. This dissertation shows some of the challenges and methods that were applied to the development of the proposed system, in addition to an overview of each of the systems that composes the equipment. The mechanical, optical, electronic and software systems are described, being each one of them detailed regarding its main features performed in the equipment. Development tools used during the design are also described. Still, the functional and performance tests and results are described, and also a comparative evaluation between the proposed system and some of the equipments currently defined as reference among physicians and users.

This master thesis deals with retinal image registration using phase correlation technique. There are described properties of retinal images and modality of scanning. A geometrical transformation encompasing scale, rotation and translation between two retinal images is considered and the whole registration framework is described. There are used retinal images from fundus camera and scanning laser ophthalmoscope (SLO). In this thesis is described corresponding bifurcations detection using phase correlation and registration using second-order polynomial transformation. The results are subjectively and objectively verificated.

The use of viable and reliable tissue preparations allows for experimental approaches in vitro that would not be possible in vivo. In this experiment, gills were isolated from gulf killifish, Fundulus grandis, and subjected to various incubation conditions. A combination of two types of media and two incubation apparatuses were compared in their ability to sustain tissues, as measured by the vital stain trypan blue (TB) and percent lactate dehydrogenase (LDH) leakage. Trypan Blue analysis indicated no significant difference in tissue viability, regardless of the treatment or incubation time. However, percent LDH leakage analysis revealed that the two most influential variables were the medium and apparatus in which the gills were incubated. Interestingly, incubation time did not have a statistically significant influence on gill viability as measured by LDH leakage. The results of LDH leakage analysis suggest that incubation using SW in the 4 ml apparatus is the best combination tested.

The retinal vasculature analysis plays an important role in the diagnosis of ophthalmological diseases, as well as general human disorders that manifest on the retina. The fundus photograph is a 2-D color image modality of the retina and is widely used in modern ophthalmology clinics due to its relatively low cost and its non-invasive access to the retina. However, due to the complexity of the retinal vasculature presented on the image and the large variation of the image quality, no automated method is able to re-construct the retinal vasculature (i.e. construct arteriovenous trees) satisfactorily, thus preventing its analysis on large-scale clinical datasets. In this thesis, we present a systematic and complete study to automatically construct the retinal vasculature on fundus photographs and apply it to a clinical dataset. First of all, a preliminary study is conducted to detect and classify important landmarks in the retinal vasculature using a machine learning method. The evaluation of this method reveals the difficulty of identifying each landmark as an independent target. Then a novel and more global method is proposed to construct retinal arteriovenous trees (A/V trees). The strategy of the proposed method is to build an over-connected vessel network, and separate it into vascular trees, then classify them into A/V trees. Particularly, by taking advantages of specific properties of the retinal vasculature, global and local information are combined together to recognize landmarks of the vasculature. Instead of recognizing each landmark independently as other methods do, this method considers the relationship between landmarks in a more global manner, thus recognizing them simultaneously and globally. With a special graph design, each landmark is associated with multiple possible configurations and costs, and a near optimal solution is selected by minimizing the costs of landmarks and the global property of the whole vascular network. With each landmark recognized, the A/V trees are easily inferred with a pixel classification method. By doing so, local noise in the images and local errors during pre-processing are corrected to some degree, and small vessels that are difficult to classify locally can also be recognized. The proposed method is compared with another method and the evaluation demonstrates its superiority. To demonstrate its potential applicability, we apply the proposed method on a cohort study data of HIV-infected patients with treatment. New metrics to analyze retinal vessel width is developed based on the A/V trees built using the proposed method, and it is compared with a conventional metric. Statistical analysis reveals the advantages of the new metric and thus indicates the benefit of the proposed method and its potential application on large datasets.

The purpose of this project is to develop a retinal imaging system suitable for routine examination or screening of mouse models that acquires fundus and Optical Coherence Tomography (OCT) images. The imaging system is composed of a digital camera with an objective for biomicroscopic examination of the fundus, an OCT interferometer, an OCT beam delivery system designed for the mouse eye, and a mouse positioning stage. The image acquisition is controlled with software that displays the fundus and OCT images in real-time, and allows the user to control the position of the OCT beam spot on the fundus image display. The system was used to image healthy mice and a mouse model of glaucoma. Fundus images and OCT scans were successfully acquired in both eyes of all mice with eyes that had clear optics. The study demonstrates the feasibility of acquiring simultaneous fundus and OCT images of the mouse retina, by a single operator, in a manner suitable for rapid evaluation of mouse models of retinal disease.

Tortuosity of eye fundus blood vessels is one of parameters that describe state of the blood vessels. It can be detected from fundus images. The increase in vessel tortuosity was observed in eyes of patients with advanced background diabetic retinopathy, papilloedema, arterial hypertension, even in some completely healthy eyes (in this case tortuosity does not change in time). Thus the feature of the line – tortousity – could be used as the diagnostic feature in medical applications. Unfortunately, completely reliable definition and numerical estimation of tortuosity of line (blood vessel) does not exist, although there were some more or less successful attempts to define it. This work presents a new way to estimate the tortuosity using the integral of square derivative of curvature. It is compared with the existing methods both theoretically and experimentally. Three types of lines have been used for experimental comparison: theoretical models of retinal vessels (straight line, parabola, sinusoid and their combinations), lines extracted from real fundus images and lines extracted from optometric scale for retinal vessel evaluation. To ease the extraction of the lines from these images a new version of one of the methods for vessel tracking has been created and implemented.