A new type of adaptive optics (AO) based on the principles of digital holography (DH) is proposed and developed for the use in wide-field and confocal retinal imaging. Digital holographic adaptive optics (DHAO) dispenses with the wavefront sensor and wavefront corrector of the conventional AO system. DH is an emergent imaging technology that gives direct numerical access to the phase of the optical field, thus allowing precise control and manipulation of the optical field. Incorporation of DH in an ophthalmic imaging system can lead to versatile imaging capabilities at substantially reduced complexity and cost of the instrument. A typical conventional AO system includes several critical hardware pieces: spatial light modulator, lenslet array, and a second CCD camera in addition to the camera for imaging. The proposed DHAO system replaces these hardware components with numerical processing for wavefront measurement and compensation of aberration through the principles of DH. We first design an image plane DHAO system which is basically simulating the process the conventional AO system and replacing the hardware pieces and complicated control procedures by DH and related numerical processing. In this original DHAO system, CCD is put at the image plane of the pupil plane of the eye lens. The image of the aberration is obtained by a digital hologram or guide star hologram. The full optical field is captured by a second digital hologram. Because CCD is not at the conjugate plane of the sample, a numerical propagation is necessary to find the image of the sample after the numerical aberration compensation at the CCD plane. The theory, simulations and experiments using an eye model have clearly demonstrated the effectiveness of the DHAO. This original DHAO system is described in Chapter 2. Different from the conventional AO system, DHAO is a coherent imaging modality which gives more access to the optical field and allows more freedom in the optical system design. In fact, CCD does not have to be put at the image plane of the CCD. This idea was first explored by testing a Fourier transform DHAO system (FTDHAO). In the FTDHAO, the CCD can directly record the amplitude point spread function (PSF) of the system, making it easier to determine the correct guide star hologram. CCD is also at the image plane of the target. The signal becomes stronger than the image plane DHAO system, especially for the phase aberration sensing. Also, the numerical propagation is not necessary. In the FTDHAO imaging system, the phase aberration at the eye pupil can be retrieved by an inverse Fourier transform (FT) of the guide star hologram and the complex amplitude of the full field optical field at the eye pupil can be obtained by an inverse FT of the full field hologram. The correction takes place at the eye pupil, instead of the CCD plane. Taking FT of the corrected field at the eye pupil, the corrected image can be obtained. The theory, simulations, and experiments on FTDHAO are detailed in chapter 3. The successful demonstration of FTDHAO encourages us to test the feasibility of putting CCD at an arbitrary diffraction plane in the DHAO system. Through theoretical formulation by use of paraxial optical theory, we developed a correction method by correlation for the general optical system to perform the DHAO. In this method, a global quadratic phase term has to be removed before the correction operation. In the formulation, it is quite surprising to find that the defocus term can be eliminated in the correlation operation. The detailed formulations, related simulations, and experimental demonstrations are presented in Chapter 4. To apply the DHAO to the confocal retinal imaging system, we first transformed the conventional line-scanning confocal imaging system into a digital form. That means each line scan is turned into a digital hologram. The complex amplitude of the optical field from each slice of the sample and aberration of the optical system can be retrieved by digital holographic process. In Chapter 5, we report our experiments on this digital line-scanning confocal imaging system. This digital line-scanning confocal image absorbs the merits of the conventional line-scanning confocal imaging system and DH. High-contrast intensity images with low coherent noise, and the optical sectioning capability are made available due to the confocality. Phase profiles of the samples become accessible thanks to DH. The quantitative phase map is even better than that from the wide field DH. We then explore the possibility of applying DHAO to this newly developed digital line-scanning confocal imaging system. Since optical field of each line scan can be achieved by the DH, the aberration contained in this field can be eliminated if we are able to obtain the phase aberration. We have demonstrated that the phase aberration can be obtained by a guide star hologram in the wide field DHAO systems. We then apply this technique to acquire the aberration at the eye pupil, remove this aberration from the optical fields of the line scans and recover the confocal image. To circumvent the effect of phase aberration on the line illumination, a small collimated laser beam is shone on the cylindrical lens. Thus the image is solely blurred by the second passage through the aberrator. This way, we can clearly demonstrate the effect of DHAO on the digital line-scanning confocal image system. Simulations and experiments are presented in chapter 6, which clearly demonstrates the validity of this idea. Since line-scanning confocal imaging system using spatially coherent light sources has proven an effective imaging tool for retinal imaging, the presented digital adaptive optics line-scanning confocal imaging system is quite promising to become a compact digital adaptive optics laser scanning confocal ophthalmoscope.

The focus of this thesis is the improvement of acquisition and processing of Mueller matrix polarimetry using a confocal scanning laser ophthalmoscope (CSLO) and the application of Mueller matrix polarimetry to image the retina. Stepper motors were incorporated into a CSLO to semi-automate Mueller matrix polarimetry and were used in retinal image acquisition. Success rates of Fourier transform based edge detection filters, designed to improve the registration of retinal images, were compared. The acquired polarimetry images were used to reassess 2 image quality enhancement techniques, Mueller matrix reconstruction (MMR) and Stokes vector reconstruction (SVR), focusing on the role of auto-contrasting or normalization within the techniques and the degree to which auto-contrasting or normalization is responsible for image quality improvement of the resulting images. Mueller matrix polarimetry was also applied to find the retardance image of a malaria infected retinal blood vessel imaged in a confocal scanning laser microscope (CSLM) to visualize hemozoin within the vessel. Image quality enhancement techniques were also applied and image quality improvement was quantified for this blood vessel. The semi-automation of Mueller matrix polarimetry yielded a significant reduction in experimental acquisition time (80%) and a non-significant reduction in registration time (44%). A larger sample size would give higher power and this result might become significant. The reduction in registration time was most likely due to less movement of the eye, particularly in terms of decreased rotation seen between registered images. Fourier transform edge detection methods increased the success rate of registration from 73.9% to 92.3%. Assessment of the 2 MMR images (max entropy and max signal-to-noise ratio (SNR)) showed that comparison to the best CSLO images (not auto-contrasted) yielded significant average image quality improvements of 158% and 4% when quantified with entropy and SNR, respectively. When compared to best auto-contrasted CSLO images, significant image quality improvements were 11% and 5% for entropy and SNR, respectively. Images constructed from auto-contrasted input images were of significantly higher quality than images reconstructed from original images. Of the 2 other images assessed (modified degree of polarization (DOPM) and the first element of the Stokes vector (S0)), DOPM and S0 yielded significant average image quality improvements quantified by entropy except for the DOPM image of the RNFL. SNR was not improved significantly when either SVR image was compared to the best CSLO images. Compared to the best auto-contrasted CSLO images, neither DOPM nor S0 improved average image quality significantly. This result might change with a larger number of participants. When MMR were applied to images of malaria infected retinal slides, image quality was improved by 19.7% and 15.3% in terms of entropy and SNR, respectively, when compared to the best CSLO image. The DOPM image yielded image quality improvements of 8.6% and -24.3% and the S0 image gave improvements of 9.5% and 9.4% in entropy and SNR, respectively. Although percent increase in image quality was reduced when images were compared to initial auto-contrasted CSLO images, the final image quality was improved when auto-contrasting occurred prior to polarimetry calculations for max SNR and max entropy images. Quantitative values of retardance could not be found due to physical constraints in the CSLM that did not allow for characterization of its polarization properties and vibrational noise. Mueller matrix polarimetry used to find the retardance image of a malaria infected retina sample did yield visualization of hemozoin within the vessel but only qualitatively. In conclusion, improvements in the acquisition and registration of CSLO images were successful in leading to considerably shorter experimentation and processing times. In terms of polarimetric image quality improvement techniques, when compared to the best CSLO image. A large proportion of the improvement was in fact due to partially or completely stretching the pixel values across the dynamic range of the images within the algorithm of each technique. However, in general the image quality was still improved by the Mueller matrix reconstruction techniques using both entropy and SNR to generate the CSLO retinal images and the CSLM imaged malaria infected sample. In the malaria sample, retinal blood vessel visualization was also qualitatively improved. The images yielded from Mueller matrix polarimetry applied to a malaria infected retinal sample localized hemozoin within the blood vessel, but a quantitative image of the phase retardance could not be achieved.

Las aberraciones ópticas determinan la formación de imágenes en el ojo, tanto en el proceso de la visión como en las observaciones oftalmoscópicas del fondo de ojo. La corrección total de estas aberraciones permitiría una resolución limitada sólo por la difracción en las pupilas utilizadas. Las aberraciones del ojo difieren de un sujeto a otro y no responden a modelos sencillos. En este trabajo se propone el uso de técnicas de Óptica Adaptativa para el desarrollo de un sistema experimental para la medida y corrección de las aberraciones estáticas del ojo. Estas técnicas pueden ser igualmente útiles para obtener imágenes de alta resolución de la retina, utilizarse en el diseño de lentes oftálmicas, etc. Para la medida de la función aberración de onda, se han utilizado dos métodos no invasivos aplicables al ojo humano: La Recuperación de Fase a partir de dos imágenes de Doble Paso, y el Sensor de Hartmann-Shack. Para la corrección de la aberración se ha utilizado un Modulador Espacial de Cristal Líquido.Se han desarrollado los procedimientos de control y de calibrado de estos métodos, y se estudia la viabilidad de aplicación para el ojo. Finalmente, se han realizado medidas de la aberración, mediante ambos métodos, y su posterior corrección mediante el modulador espacial de cristal líquido, en un ojo artificial y en sujetos reales. / The image formation properties of the eye are determined by the aberrations of the optics. The complete correction of the aberrations would allow diffraction-limited resolution. The aberrations of the eye are not easily modeled and are different for each subject.This thesis proposes the use of adaptive optics techniques to measure and correct the static aberrations of the eye. The principles and methods developed are useful in specific applications, i.e., high-resolution retinal imaging, ophthalmic lens design, etc.Two non-invasive methods have been used to measure the wave aberration function: Phase Retrieval Techniques from two double-pass retinal images; and the Hartmann-Shack sensor. A Liquid Crystal Spatial Light Modulator was used to adaptively correct the wave front aberration of the eye.This thesis also includes guidelines to calibrate and control the proposed techniques.Finally, experimental explorations of these methods are reported. Several results are presented, including the measure and the subsequent compensation of the wave aberration for artificial and human eyes.

The focus of this thesis is the improvement of acquisition and processing of Mueller matrix polarimetry using a confocal scanning laser ophthalmoscope (CSLO) and the application of Mueller matrix polarimetry to image the retina. Stepper motors were incorporated into a CSLO to semi-automate Mueller matrix polarimetry and were used in retinal image acquisition. Success rates of Fourier transform based edge detection filters, designed to improve the registration of retinal images, were compared. The acquired polarimetry images were used to reassess 2 image quality enhancement techniques, Mueller matrix reconstruction (MMR) and Stokes vector reconstruction (SVR), focusing on the role of auto-contrasting or normalization within the techniques and the degree to which auto-contrasting or normalization is responsible for image quality improvement of the resulting images. Mueller matrix polarimetry was also applied to find the retardance image of a malaria infected retinal blood vessel imaged in a confocal scanning laser microscope (CSLM) to visualize hemozoin within the vessel. Image quality enhancement techniques were also applied and image quality improvement was quantified for this blood vessel. The semi-automation of Mueller matrix polarimetry yielded a significant reduction in experimental acquisition time (80%) and a non-significant reduction in registration time (44%). A larger sample size would give higher power and this result might become significant. The reduction in registration time was most likely due to less movement of the eye, particularly in terms of decreased rotation seen between registered images. Fourier transform edge detection methods increased the success rate of registration from 73.9% to 92.3%. Assessment of the 2 MMR images (max entropy and max signal-to-noise ratio (SNR)) showed that comparison to the best CSLO images (not auto-contrasted) yielded significant average image quality improvements of 158% and 4% when quantified with entropy and SNR, respectively. When compared to best auto-contrasted CSLO images, significant image quality improvements were 11% and 5% for entropy and SNR, respectively. Images constructed from auto-contrasted input images were of significantly higher quality than images reconstructed from original images. Of the 2 other images assessed (modified degree of polarization (DOPM) and the first element of the Stokes vector (S0)), DOPM and S0 yielded significant average image quality improvements quantified by entropy except for the DOPM image of the RNFL. SNR was not improved significantly when either SVR image was compared to the best CSLO images. Compared to the best auto-contrasted CSLO images, neither DOPM nor S0 improved average image quality significantly. This result might change with a larger number of participants. When MMR were applied to images of malaria infected retinal slides, image quality was improved by 19.7% and 15.3% in terms of entropy and SNR, respectively, when compared to the best CSLO image. The DOPM image yielded image quality improvements of 8.6% and -24.3% and the S0 image gave improvements of 9.5% and 9.4% in entropy and SNR, respectively. Although percent increase in image quality was reduced when images were compared to initial auto-contrasted CSLO images, the final image quality was improved when auto-contrasting occurred prior to polarimetry calculations for max SNR and max entropy images. Quantitative values of retardance could not be found due to physical constraints in the CSLM that did not allow for characterization of its polarization properties and vibrational noise. Mueller matrix polarimetry used to find the retardance image of a malaria infected retina sample did yield visualization of hemozoin within the vessel but only qualitatively. In conclusion, improvements in the acquisition and registration of CSLO images were successful in leading to considerably shorter experimentation and processing times. In terms of polarimetric image quality improvement techniques, when compared to the best CSLO image. A large proportion of the improvement was in fact due to partially or completely stretching the pixel values across the dynamic range of the images within the algorithm of each technique. However, in general the image quality was still improved by the Mueller matrix reconstruction techniques using both entropy and SNR to generate the CSLO retinal images and the CSLM imaged malaria infected sample. In the malaria sample, retinal blood vessel visualization was also qualitatively improved. The images yielded from Mueller matrix polarimetry applied to a malaria infected retinal sample localized hemozoin within the blood vessel, but a quantitative image of the phase retardance could not be achieved.

This diploma thesis is dealing with the detection of blood vessels pulsation in retinal sequences. The goal is to create an algorithm for objective evaluation of pulsation in retinal video sequences.

Retinal imaging enables the visualization of a portion of the human microvasculature in-vivo and non-invasively. The scanning laser ophthalmoscope (SLO), provides images characterized by an ultra-wide field of view (UWFoV) covering approximately 180-200º in a single scan, minimizing the discomfort for the subject. The microvasculature visible in retinal images and its changes have been vastly investigated as candidate biomarkers for different types of systemic conditions like cardiovascular disease (CVD), which currently remains the main cause of death in Europe. For the CARMEN study, UWFoV SLO images were acquired from more than 1,000 people who were recruited from two studies, TASCFORCE and SCOT-HEART, focused on CVD. This thesis presents an automated system for SLO image processing and computation of candidate biomarkers to be associated with cardiovascular risk and MRI imaging data. A vessel segmentation technique was developed by making use of a bank of multi-scale matched filters and a neural network classifier. The technique was devised to minimize errors in vessel width estimation, in order to ensure the reliability of width measures obtained from the vessel maps. After a step of refinement of the centrelines, a multi-level classification technique was deployed to label all vessel segments as arterioles or venules. The method exploited a set of pixel-level features for local classification and a novel formulation for a graph cut approach to partition consistently the retinal vasculature that was modelled as an undirected graph. Once all the vessels were labelled, a tree representation was adopted for each vessel and its branches to fully automate the process of biomarker extraction. Finally, a set of 75 retinal parameters, including information provided by the periphery of the retina, was created for each image and used for the biomarker investigation.

Abstract Glaucoma is a group of eye diseases characterized by a chronic, progressive optic neuropathy. During the disease process, the axon damage of the retinal ganglion cells leads to changes in the retinal nerve fiber layer, causing optic nerve head, and visual field defects typical of glaucoma. The Heidelberg Retina Tomograph (HRT) is a confocal scanning laser imaging device acquiring and analysing three-dimensional data of the ocular fundus wit good accuracy and reproducibility. Conventional planimetric measurements were compared to those taken with the HRT in a pilot study of 12 eyes with early glaucomatous optic disc, retinal nerve fiber layer and/or visual field abnormalities. The neuroretinal rim area measurements and cup-to-disc area ratio did not differ statistically from each other when using these two different methods. The effect of four different reference levels on the HRT parameter measurement values was tested in two separate studies. In the first study there were 67 eyes, 40 of the eyes were healthy and 27 eyes had glaucoma of different stages. Then, 279 eyes, 180 of which were non-glaucomatous and 99 glaucoma eyes, were included in another study. The flexible reference level gave the most reliable HRT parameter measurement values in both non-glaucomatous and glaucomatous eyes. The ability of the HRT parameters to separate between non-glaucomatous and glaucomatous eyes was tested in 77 eyes, 40 of the eyes were non-glaucomatous, 10 ocular hypertensives and 27 eyes had different stages of glaucoma. The reference level dependent HRT parameters cup-to-disc area ratio, vertical linear cup-to-disc ratio, mean retinal nerve fiber layer thickness (RNFLt) and rim volume as well as the reference level non-dependent HRT parameter, cup shape measure (CSM), separated best between the clinical groups. The best combination of the HRT and other structural and functional parameters in separating between non-glaucomatous and glaucomatous eyes was studied in 55 eyes. There were 32 non-glaucomatous eyes and 23 eyes with ocular hypertension or glaucoma. CSM, RNFLt, together with age- and lens coloration-corrected mean deviation of the B/Y perimetry showed good discrimination (ROC area 0.91) between non-glaucomatous and glaucomatous eyes. / Tiivistelmä Glaukooma koostuu joukosta hitaasti eteneviä näköhermon rappeumasairauksia. Sairausprosessin aikana verkkokalvon gangliosolujen aksonivaurio johtaa muutoksiin verkkokalvon hermosäiekerroksessa ja näköhermon päässä aiheuttaen glaukoomalle tyypillisiä näkökenttämuutoksia. The Heidelberg Retina Tomograph (HRT) on konfokaali laserskanneritekniikkaan perustuva kuvantamislaite, joka tuottaa ja analysoi silmänpohjasta saatua kolmiulotteista mittaustietoa tarkasti ja toistettavasti. Tavanomaisen planimetrian antamia mittaustuloksia verrattiin HRT:n antamiin tuloksiin 12:ssa silmässä, joissa oli todettu varhaisia glaukoomamuutoksia. Näköhermon pään hermoreunan (rim) pinta-ala ja keskuskuopan suhde papillan läpimittaan eivät poikenneet tilastollisesti toisistaan näitä kahta menetelmää käytettäessä. Neljän eri referenssitason vaikutusta HRT-parametrien mittausarvoihin testattiin kahdessa eri tutkimuksessa. Ensimmäisen tutkimusaineisto koostui yhteensä 67:stä silmästä, joista 40 oli terveitä ja 27:ssä eriasteisia glaukoomamuutoksia. Toisessa tutkimuksessa oli yhteensä 279 silmää, joista 180 oli terveitä ja 99:llä oli glaukooma. Papillomakulaarisäikeisiin tukeutuva, fleksiibeli referenssitaso antoi luotettavimmat HRT-parametrien mittaustulokset sekä terveissä että glaukoomasilmissä. HRT-parametrien kykyä erottaa terveet silmät glaukomatoottisista testattiin yhteensä 77:ssä silmässä, joista 40 oli terveitä, 10 oli korkeapaineisia ilman glaukoomamuutoksia, ja 27:ssä oli glaukoomamuutoksia. Referenssitasosta riippuvaiset HRT-parametrit, keskuskuopan suhde papillan läpimittaan, vertikaali-lineaarinen keskuskuopan suhde papillan läpimittaan, keskimääräinen verkkokalvon hermosäiekerroksen paksuus (RNFLt) ja `rim´:in tilavuus samoin kuin referenssitasosta riippumaton keskuskuopan ´vinous´-mitta (CSM) erottelivat parhaiten nämä kliiniset ryhmät toisistaan. Terveitä ja glaukoomasilmiä erottelevaa HRT:n ja muiden rakenteellisten ja toiminnallisten parametrien kombinaatiota etsittiin 55:n silmän aineistosta. Silmistä 32 oli terveitä ja 23 korkeapaineisia ja/tai glaukoomavaurioisia. CSM ja RNFLt, yhdessä iän ja mykiövärjäytymisen suhteen korjatun sinikeltaperimetrian keskipoikkeaman kanssa osoittivat hyvää erottelukykyä (ROC area 0.91) terveiden ja glaukoomasilmien välillä.

This paper deals with identification of persons using retinal biometry. The retinal vasculature is invariant and unique to everyone, which determines it for biometric purposes. The first part of the work includes information about biometrics, biometric systems and reliability measures. The next part describes the principle of using experimental video ophthalmoscope, which was used for retinal vascular imaging and includes the literature research of use of retinal images for biometrics, feature extraction methods and similarity measures. Finally, two algorithms to use the input data are proposed and realized in programming environment MATLAB®. The methods are tested and evaluated on a data set from experimental video ophthalmoscope and on publicly available STRaDe and DRIVE databases.

This master´s thesis deals with blood vessel segmentation in retinal image data. The theoretical part is focused on the basic description of anatomy and physiology of the eye and methods of observing the back of the eye. This thesis also describes the principles of classical and convolutional neural networks and segmentation techniques that are used to segment blood vessel in retinal images. In the practical part, a segmentation method using convolutional neural network U-net is implemented. This neural network is trained on the three datasets. Two datasets include images from experimental video ophthalmoscope. Because it impossible to compare the results of these two datasets with any other methods of retinal blood vessel segmentation, U-net is trained on other dataset that is HRF database. This dataset includes fundus images. The results of testing on this dataset serves for comparing results with other methods of retinal blood vessel segmentation.

Studies demonstrate neuro-retinal damage in patients with diabetes and no clinically visible diabetic retinopathy. It is unknown which retinal regions are most vulnerable to diabetes. We hypothesized that the standard and slow-flash (sf-) multifocal electroretinogram (mfERG) and adaptive optics (AO) imaging will localize retinal regions of vulnerability. Fifty-five adolescents with diabetes and 54 controls underwent mfERG testing to isolate predominately retinal bipolar cell activity and sf-mfERG testing to isolate three oscillatory potentials (OPs) from intraretinal amacrine and interplexiform cells. Greatest mfERG delays were in the superior temporal quadrant and at 5°-10° eccentricity. Greatest sf-mfERG delays were found at different eccentricities for each OP. Twenty adolescents with diabetes and 14 controls underwent AO imaging. No significant differences in cone photoreceptor density were found; however, patients showed a trend towards reduced density in the superior nasal region. Inner retinal structures may be more susceptible to damage by diabetes than outer retinal structures.