Automatic Screening and Classification of Diabetic Retinopathy Eye Fundus Image

Diabetic Retinopathy (DR) is a disorder of the retinal vasculature. It develops to some degree in nearly all patients with long-standing diabetes mellitus and can result in blindness. Screening of DR is essential for both early detection and early treatment. This thesis aims to investigate automatic methods for diabetic retinopathy detection and subsequently develop an effective system for the detection and screening of diabetic retinopathy. The presented diabetic retinopathy research involves three development stages. Firstly, the thesis presents the development of a preliminary classification and screening system for diabetic retinopathy using eye fundus images. The research will then focus on the detection of the earliest signs of diabetic retinopathy, which are the microaneurysms. The detection of microaneurysms at an early stage is vital and is the first step in preventing diabetic retinopathy. Finally, the thesis will present decision support systems for the detection of diabetic retinopathy and maculopathy in eye fundus images. The detection of maculopathy, which are yellow lesions near the macula, is essential as it will eventually cause the loss of vision if the affected macula is not treated in time. An accurate retinal screening, therefore, is required to assist the retinal screeners to classify the retinal images effectively. Highly efficient and accurate image processing techniques must thus be used in order to produce an effective screening of diabetic retinopathy. In addition to the proposed diabetic retinopathy detection systems, this thesis will present a new dataset, and will highlight the dataset collection, the expert diagnosis process and the advantages of the new dataset, compared to other public eye fundus images datasets available. The new dataset will be useful to researchers and practitioners working in the retinal imaging area and would widely encourage comparative studies in the field of diabetic retinopathy research. It is envisaged that the proposed decision support system for clinical screening would greatly contribute to and assist the management and the detection of diabetic retinopathy. It is also hoped that the developed automatic detection techniques will assist clinicians to diagnose diabetic retinopathy at an early stage

Advancing Hyperacuity for Vision Screening

Introduction: The human visual system is extremely sensitive to certain spatial visual tasks. It can detect the subtle misalignment of closer objects to a degree of 2-5 arcseconds, which is smaller than the foveal cone diameter or spacing. This ability is referred to as hyperacuity, and one such visual task is the Vernier task, which involves misalignment detection of Vernier lines or dots. It is also called Vernier acuity and has a significant diagnostic value for screening various eye abnormalities. However, due to methodological and technical limitations, its utility was restricted to laboratory applications due to concerns over test reliability and testing time. I hypothesized that applying advanced psychophysical procedures, techniques, and modern technological interventions might improve the Vernier acuity testing standards for clinical consideration. Therefore, I attempted to address the challenges noticed in the literature by advancing the methodology and technicality to improve the Vernier acuity test efficiency for clinical application. Aims: ⸰ Experiment 1 (Chapter-2): To develop a software application and assess the Vernier acuity program performance, measurements, and stimuli characteristics. ⸰ Experiment 2 (Chapter-3): To enhance the Vernier acuity program efficiency, assess program performance and reliability for technical validation. ⸰ Experiment 3 (Chapter-4): To modify the Vernier acuity program for the visual field quantification (Hyperacuity perimetry) and assess reliability for technical validation. ⸰ Experiment 4 (Chapter-5): To develop a software application for the visual distortions quantification (Metamorphopsia) using Vernier acuity-based bisectional program and assess reliability for technical validation. Methods: This study was performed through two pilot studies: the first pilot study had five adult volunteers with the best corrected visual acuity of 20/20 vision in the right eye (tested right eye only) and included all the experiments from Chapter 2, whereas the second pilot study was carried out on 21 adult emmetrope (unaided 20/20 vision) volunteers (tested both eyes individually) and included all the experiments from Chapters-3, 4, and 5. I used PsychoPy3 to develop each software program. However, I employed two methods to provide the test results efficiently and reliably for clinical testing. I developed three software applications and could only perform technical validation because of the pandemic. ⸰ Experiment 1 (Chapter-2): I programmed a software program and employed a 3-Down, 1-Up adaptive staircase method and three alternative forced choices technique to quantify the Vernier acuity. The Vernier acuity was measured at seven vertical separations (gaps) to assess test performance. The initial testing was focused on determining test performance using stimuli shapes, followed by technical validation of the software application program and assessment of stimuli contrast for standardization. ⸰ Experiment 2 (Chapter-3): I adjusted the measurement parameters to improve test efficiency. I assessed the program performance from response accuracy, reaction time, and testing time, along with repeatability of measurements for technical validation of the Vernier acuity program. ⸰ Experiment 3 (Chapter-4): I modified the Vernier acuity program to quantify the hyperacuity perimetry in superior, inferior, nasal, and temporal visual fields. I assessed the repeatability of measurements for technical validation of this modified Vernier acuity program. ⸰ Experiment 4 (Chapter-5): I programmed a Vernier bisection program to quantify the metamorphopsia using a method of adjustment. The testing involved Vernier stimuli in two different orientations and referred to them as patterns (A and B), Pattern-A had a presentation of two vertical and horizontal line stimuli that are equally away from the center of the screen in either direction, whereas pattern B involved presentations of pattern-A at oblique angles to the screen center. Using both patterns, I measured the metamorphopsia in central 5 degrees and assessed the repeatability of the measurements and testing time for technical validation of the Vernier bisection program. Results: ⸰ Experiment 1 (Chapter-2): In the initial experiment, the line stimuli achieved comparable measurements to dot stimuli at most gap sizes except at 32 arcminutes of gap size. The test detected the lowest misalignment of 2 arcseconds at 2 arcminutes of gap size. The mean lowest acuity was below 8 arcseconds at 2 arcminutes, and the highest acuity was within an arcminute at 128 arcminutes. The negative contrast line stimuli were comparably precise to positive line stimuli at most gap sizes except at 16 arcminutes. ⸰ Experiment 2 (Chapter-3): The right eyes were repeatable at most of the gap sizes except for the 32 and 64 gap sizes, whereas the left eyes were repeatable at all gap sizes except 128. The right and left eye measurements were statistically the same at both visits. Since no difference was observed between the eyes, results from both eyes were compiled to assess the test performance and response accuracy. The Vernier acuity measured at 16, 8, 4, and 2 gap sizes were statistically repeatable, and the correlation was positive but weak. The response accuracy was estimated to be above 90% for mean correct responses, below 3% for mean incorrect responses, and about 5% for mean aligned responses through the gap sizes at both visits. The estimated reaction time was just below a second for mean correct responses, below 0.75 seconds for the mean incorrect responses, and about 2.5 seconds for the mean aligned responses. The test time was below 2 minutes at each gap size at both visits. ⸰ Experiment 3 (Chapter-4): The right eye hyperacuity perimetry results were repeatable in all four quadrants of 15 gap size, and the correlation was positive but weak in all quadrants except the superior visual field, where the correlation was negative and weak. Whereas for gap size 30, the results were repeatable in all quadrants except the inferior visual field, where the results were not repeatable. However, results from all the quadrants had a positive but weak correlation. The left eye hyperacuity perimetry results were repeatable in all quadrants of 15 gap size, except the nasal visual field, where the results were not repeatable. However, results from all the quadrants had a positive but weak correlation. For gap size 30, the results were repeatable in all four quadrants, and all quadrants had a positive but weak correlation. The results from both eyes showed no significant difference for a gap of 15 arcminutes. However, there was a substantial difference between the eyes only at the inferior field for a gap of 30 arcminutes. ⸰ Experiment 4 (Chapter-5): The right eye metamorphopsia results were repeatable in the central 5 degrees using a pattern A with a positive but weak correlation at all degrees except 5 and 1 degrees, where the correlation was negative and weak. Similarly, the results were repeatable in the central 5 degrees using a pattern B except for 5 degrees, and the correlation was positive but weak at all degrees except 3 and 2 degrees, where there was a negative and weak correlation. On the other hand, the left eye results were repeatable in the central 5 degrees using a pattern A with positive and moderate to strong correlations at all degrees. Similarly, the results were repeatable in the central 5 degrees using a pattern B except for 4 degrees, and the correlation was positive and moderate to strong at all degrees. There was no difference between the eyes for individual patterns at both visits. Conclusions: ⸰ Experiment 1 (Chapter-2): The developed software application program measured the Vernier acuity precisely using 3-down, 1-up, an adaptive staircase method, and the 3AFC technique. In addition, I standardized stimuli for shape and contrast to measure the Vernier acuity. The calculated results are precise and consistent with the previously reported data. Therefore, motivating to advance the test further for Vernier acuity testing. While performing the test, some areas for improvement were identified. By adjusting the necessary parameters, the test's efficiency can be enhanced. I plan to make those adjustments in the following pilot testing and perform technical validation. ⸰ Experiment 2 (Chapter-3): I adjusted the necessary parameters to improve the efficiency of the Vernier acuity testing. The results showed that the program is efficient, robust, and repeatable. The mean Vernier acuity measured at seven different gap sizes was consistent with previously reported data and comparable with pilot study 1 results. This cohort’s Vernier acuity measured at smaller gap sizes was highly dependable. The measurements were significantly repeatable at most gap sizes but were poorly dependable. Therefore, this program may need further modifications to achieve better reliable results for clinical testing. ⸰ Experiment 3 (Chapter-4): I modified the Vernier acuity program to quantify the Vernier acuity in eccentric 5 degrees of the macula (para-foveal area). The right eye results were repeatable for 15 arc minutes of gap size, whereas the left eye results were repeatable for the 30 arc minutes of gap size. However, both eyes had poor reliability at most of the gap sizes except the superior field of gap 30, where the reliability was moderate. This inconsistency between the eyes could be due to distinct reasons and therefore needs further investigations to address the underlying cause. ⸰ Experiment 4 (Chapter-5): I programmed a software application to quantify the metamorphopsia using a method of adjustment. The right eye results were repeatable at most gap sizes for both patterns. However, the measurements were poorly dependable at most gap sizes of both patterns. Similarly, the left eye results were repeatable at most gap sizes for both patterns. However, the measurements were poorly dependable at most gap sizes of both patterns, except at 3 degrees of pattern A and 5 and 2 degrees of pattern B, where the reliability was moderate. Further modifications in the program may provide better reliability for clinical testing

Biomedical Engineering

Biomedical engineering is currently relatively wide scientific area which has been constantly bringing innovations with an objective to support and improve all areas of medicine such as therapy, diagnostics and rehabilitation. It holds a strong position also in natural and biological sciences. In the terms of application, biomedical engineering is present at almost all technical universities where some of them are targeted for the research and development in this area. The presented book brings chosen outputs and results of research and development tasks, often supported by important world or European framework programs or grant agencies. The knowledge and findings from the area of biomaterials, bioelectronics, bioinformatics, biomedical devices and tools or computer support in the processes of diagnostics and therapy are defined in a way that they bring both basic information to a reader and also specific outputs with a possible further use in research and development

Multimodal imaging in age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness and affects approximately one in seven Australians aged 50 years and above. Currently, this complex condition is not easily and uniformly assessed. The signs of AMD differ between eyes and also occur in other macular disorders. This hinders accurate diagnosis and classification, which is fundamental to optimal patient care. Ocular imaging and visual function assessment have the potential to minimise the devastating consequences of disease through early detection. However, multiple devices are now commercially available and the impact of these technologies in clinical practice may not be straightforward. For instance, their usefulness may depend on accessibility and the operator’s knowledge and clinical skills. The impact on patient management, as well as alternative models of eye-care delivery, requires clarification. This thesis aims to explore the current and potential utility of imaging technologies (optical coherence tomography, infrared imaging, monochromatic retinal photography and fundus autofluorescence) in the assessment and management of AMD and other diseases of retinal pigment epithelium dysfunction. The findings show that optometrists self-describe high levels of practice competency and make ready use of imaging in everyday practice. However, they also unwittingly demonstrated low awareness of the evidence base in AMD. Furthermore, when their interpretation of images was tested using a series of case vignettes, their diagnostic accuracy as a group improved by only five per cent (from 61 per cent to 66 per cent); their tendency to refer increased by four per cent. These factors might be improved through education. A series of open-access, chair-side reference charts were consequently devised to help optometrists use imaging technologies more effectively in clinical practice. The additive contribution of multimodal structural and functional testing was particularly emphasised. Finally, a novel model of intermediate-tier eye-care in Australia was shown to substantially reduce the number of false positive cases or cases without a specific diagnosis. Interestingly, this model was acclaimed by reviewers as “scoring highly for originality and of international relevance”. Most excitingly, the thesis concludes with future directions regarding collaborative care and multimodal imaging, where detection of disease might be facilitated via a computational approach

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.Siirretty Doriast

How microplastics and adsorbed pollutants affect zebrafish development?

A presença de microplásticos no ecossistema aquático é, nos dias de hoje, uma realidade extremamente preocupante que acarreta sérios riscos para o meio ambiente e para a saúde pública. A capacidade dos microplásticos de adsorverem poluentes orgânicos, como é o caso do benzo[α]pireno (BaP), levanta preocupações adicionais, pois cria uma nova rota de entrada de compostos tóxicos na cadeia alimentar. No entanto, o conhecimento atual sobre o impacto dos microplásticos, inalterados e/ou contaminados, nos organismos aquáticos é ainda insuficiente e requer estudos adicionais. O trabalho desenvolvido no âmbito desta tese pretende assim fornecer novos dados sobre os efeitos biológicos dos microplásticos utilizando como modelo o peixe-zebra (Danio rerio). Pequenos teleósteos, como é o caso do peixe-zebra, oferecem vantagens significativas relativamente aos modelos animais clássicos e são atualmente utilizados como organismos de primeira linha para avaliar os riscos ambientais associados aos compostos tóxicos presentes nos meios aquáticos. Estudos toxicológicos requerem o uso de materiais inertes e condições controladas, todavia, nenhum dos sistemas atualmente comercializados é adequado para avaliar o efeito tóxico dos microplásticos. Estes sistemas contêm componentes feitos de polímeros plásticos que podem libertar partículas plásticas micrométricas, lixiviar os constituintes químicos ou adsorver os compostos químicos em estudo. O sistema de aquários autônomo ZEB316 foi desenvolvido no âmbito deste trabalho com o objetivo de suprimir esta necessidade e de facultar uma solução económica e fácil de implementar que permita a realização de estudos toxicológicos de última geração. Este sistema é construído com materiais inertes e resistentes à corrosão e proporciona boas condições de alojamento através de um sistema eficiente de recirculação e filtragem da água. A avaliação dos parâmetros da água e do desempenho do crescimento dos peixes mostrou que o sistema ZEB316 oferece condições de alojamento comparáveis à dos sistemas comercialmente disponíveis. A maioria dos resultados obtidos no âmbito desta dissertação provêm da análise de imagens de microscopia de campo claro e/ou fluorescência. Embora o uso de imagens de microscopia seja comum na maioria dos laboratórios e permita obter uma quantidade considerável de informação, a análise destas imagens é geralmente um processo moroso, em parte devido à falta de ferramentas automáticas/semiautomáticas dedicadas que permitam a sua análise. Nesse sentido, desenvolvemos diversas macros para o software ImageJ, com o objetivo de reduzir o erro associado à análise pelo usuário, aumentando assim a reprodutibilidade dos dados e a padronização das metodologias experimentais. Uma vez que o foco deste trabalho está centrado no estudo da osteotoxicidade, é de salientar o desenvolvimento de um conjunto de macros específicas para esse fim e denominadas ZFBONE. Este conjunto de ferramentas permite aos usuários avaliar, a partir de imagens 2D, parâmetros morfométricos de várias estruturas ósseas (por exemplo, opérculo, raios e escamas da barbatana caudal), mas também a extensão e a intensidade das colorações específicas do osso. Além disso, outras macros foram desenvolvidas para outros fins, por exemplo, para analisar os vários parâmetros morfométricos relativos aos embriões de peixe ou para avaliar cortes histológicos. Uma vez desenvolvidos os meios e técnicas necessários para a execução dos ensaios toxicológicos, e subsequente análise dos resultados, procedeu-se à realização das várias experiências que visam compreender efeito biológico dos microplásticos e contaminates no peixe-zebra. Assim, larvas de peixe-zebra foram produzidas e mantidas no sistema previamente descrito, ZEB316, e expostas cronicamente, durante o seu desenvolvimento, a partículas de polietileno de 20- 27 μm, inalteradas (MP) ou contaminadas com benzo[α]pireno (MP-BaP). Estas partículas foram adicionadas à dieta dos peixes através de uma suplementação a 1% m/m. Apesar de não se ter registado qualquer alteração ao nível dos parâmetros morfológicos aos 30 dias pós-fertilização (dpf), a presença de MP e MP-BaP acabou por ter um efeito negativo no crescimento dos espécimes aos 90 e 360 dpf. A fecundidade relativa, a morfologia do ovo/embrião e a área do vitelo também sofreram um impacto negativo em peixes-zebra alimentados com MP-BaP. No que respeita ao estado geral do esqueleto, os peixes expostos a dietas experimentais contendo MP e MP-BaP sofreram um aumento significativo na incidência de deformações esqueléticas aos 30 dpf quando comparados com peixes alimentados com a dieta controlo, bem como um desenvolvimento anómalo da barbatana caudal e escamas, e uma diminuição da qualidade do osso aos 90 dpf. Um comprometimento da formação óssea intergeracional foi também observado na prole de espécimes expostos a MP ou MP-BaP, que se refletiu numa redução do osso opercular dos descendentes aos 6 dpf. Além de um claro efeito no desenvolvimento ósseo, a análise histológica do intestino revelou ainda um número reduzido de células caliciformes em peixes alimentados com dieta MP-BaP, um claro sinal de inflamação intestinal. Finalmente, a exposição das larvas a MP-BaP levou a um aumento da expressão de genes associados à via de resposta do BaP, ao mesmo tempo que impactou negativamente na expressão de genes envolvidos no estresse oxidativo. Os resultados obtidos indicaram um maior comprometimento do desenvolvimento ósseo no peixe-zebra quando sujeito a uma dieta contendo microplásticos contaminados com BaP (MP-BaP), por comparação com a dieta contendo microplásticos não contaminados (MP). Outros autores demostraram também que a presença de BaP afeta a formação das vértebras e o desenvolvimento generalizado do esqueleto, no entanto os mecanismos envolvidos nestes fenómenos permanecem pouco estudados. Desta forma, realizámos ensaios adicionais in vivo com o objetivo de avaliar os efeitos osteotóxicos do BaP durante o desenvolvimento e regeneração óssea em peixe-zebra. A exposição aguda de larvas de peixe-zebra entre os 3 e os 6 dpf ao BaP levou a uma redução do tamanho do osso opercular e uma diminuição quantitativa de células positivas para osteocalcina, indicando um efeito composto na maturação dos osteoblastos. Por sua vez, quando se trata de uma exposição crônica das larvas de peixe-zebra ao BaP, entre os 3 e os 30 dpf, verificou-se que o desenvolvimento do esqueleto axial é afetado, aumentando a incidência e gravidade das deformações esqueléticas. Em peixes jovens adultos, observou-se ainda que a exposição ao BaP afetou não só a mineralização dos raios da barbatana caudal e escamas recém-formados, como prejudicou também o seu padrão morfológico, fenómenos que têm por base uma remodelação óssea desequilibrada. Relativamente às análises de expressão genética de vários marcadores verificou-se que o BaP induziu a ativação das vias xenobióticas e metabólicas e impactou negativamente na formação e organização da matriz extracelular. Curiosamente, a exposição ao BaP regulou positivamente os marcadores de inflamação nas larvas e aumentou o recrutamento de neutrófilos. Uma interação direta entre neutrófilos e a matriz extracelular óssea, ou células responsáveis pela formação do osso, foi observada in vivo, o que sugere um papel dos neutrófilos nos mecanismos subjacentes à osteotoxicidade do BaP. Globalmente, os resultados obtidos no âmbito deste trabalho sugerem que a exposição crónica a microplásticos inalterados e/ou contaminados não prejudica apenas o crescimento dos peixes, mas também o seu desempenho a nível reprodutivo, saúde geral do seu esqueleto, e compromete a descendência por meio de efeitos intergeracionais. Além disso, este trabalho fornece novos dados sobre os principais mecanismos celulares e moleculares envolvidos na osteotoxicidade do BaP e aborda o possível papel dos neutrófilos na redução da resposta óssea inflamatória.The presence of microplastics in the aquatic ecosystem represents a major issue for the environment and human health. The capacity of organic pollutants such as benzo[α]pyrene (BaP) to adsorb onto microplastic particles raises additional concerns, as it creates a new route for toxic compounds to enter the food web. Current knowledge on the impact of pristine and/or contaminated microplastics on aquatic organisms remains insufficient, and this work aims at providing new insights by evaluating their biological effects in zebrafish (Danio rerio). The ZEB316, a standalone housing system built with inert materials, and a comprehensive set of ImageJ semi-automatic tools were first developed and optimized to perform state-of-the-art toxicological studies and obtain meaningful data from morphometric analysis of brightfield/ fluorescence images. Zebrafish larvae were exposed throughout their development to polyethylene microplastics, pristine or spiked with BaP, supplemented in the fish diet. While exposure up to 30 days post-fertilization only increased the incidence of skeletal deformities, long-term exposure to pristine/contaminated microplastics not only jeopardized fish growth, reproduction performance, and skeletal health, but it also caused an intergenerational effect. To further study the mechanisms underlying BaP osteotoxicity, several bone-related in vivo assays were used to evaluate the effects of waterborne exposure to BaP during bone development and regeneration. BaP inhibited osteoblast maturation and ECM mineralization and stimulated osteoclast activity, thus affecting bone remodeling. Transgenic and transcriptomic approaches suggested that besides the activation of xenobiotic and metabolic pathways, which may negatively impact extracellular matrix formation and organization, BaP activates inflammatory mechanisms that recruit neutrophils, which affect both osteoblast and osteoclast activity, possibly through a direct interaction of the neutrophils with the bone matrix. This work provides novel data on the effects of microplastics exposure during zebrafish development, in particular its osteotoxic effects, and gives new insights into the cellular and molecular players involved in BaP osteotoxicity.Marco Tarasco was supported by the Portuguese Foundation for Science and Technology (FCT) through the PhD grant SFRH/BD/128634/2017 and COVID/BD/151848/2021 and by NEUBIAS-COST STSM program as part of action CA15124. This work was funded by FCT through projects UID/Multi/04326/2019, UID/00350/2020, UIDB/04326/2020, UID/MAR/00350/2013 and from the operational programs MAR2020 and COMPETE 2020 through projects OSTEOMAR MAR-02.01.01-FEAMP-0057 and EMBRC.PT ALG-01-0145-FEDER-022121

Optical microscopy to study the role of cytoskeleton in cell locomotion and virus trafficking

3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1 . The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF...3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1 . The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Abstracts of manuscripts submitted in 1991 for publication

This volume contains the abstracts of manuscripts submitted for publication during calendar year 1991 by the staff and students of the Woods Hole Oceanographic Institution. We identify the journal of those manuscripts which are in press or have been published. The volume is intended to be informative, but not a bibliography. The abstracts are listed by title in the Table of Contents and are grouped into one of our five departents, Marine Policy Center, Coastal Research Center, or the student category. An author index is presented in the back to facilitate locating specific papers