Digital holography is a technique for the recording of holograms via CCD/CMOS devices and enables their subsequent numerical reconstruction within computers, thus avoiding the photographic processes that are used in optical holography. This thesis investigates the various techniques which have been developed for digital holography. It develops and successfully demonstrates a number of refinements and additions in order to enhance the performance of the method and extend its applicability. The thesis contributes to both the experimental and numerical analysis aspects of digital holography. Regarding experimental work: the thesis includes a comprehensive review and critique of the experimental arrangements used by other workers and actually implements and investigates a number of these in order to compare performance. Enhancements to these existing methods are proposed, and new methods developed, aimed at addressing some of the perceived short-comings of the method. Regarding the experimental aspects, the thesis specifically develops:• Super-resolution methods, introduced in order to restore the spatial frequencies that are lost or degraded during the hologram recording process, a problem which is caused by the limited resolution of CCD/CMOS devices.• Arrangements for combating problems in digital holography such as: dominance of the zero order term, the twin image problem and excessive speckle noise.• Fibre-based systems linked to tunable lasers, including a comprehensive analysis of the effects of: signal attenuation, noise and laser instability within such systems.• Two-source arrangements for contouring, including investigating the limitations on achievable accuracy with such systems. Regarding the numerical processing, the thesis focuses on three main areas. Firstly, the numerical calculation of the Fresnel-Kirchhoff integral, which is of vital importance in performing the numerical reconstruction of digital holograms. The Fresnel approximation and the convolution approach are the two most common methods used to perform numerical reconstruction. The results produced by these two methods for both simulated holograms and real holograms, created using our experimental systems, are presented and discussed. Secondly, the problems of the zero order term, twin image and speckle noise are tackled from a numerical processing point of view, complementing the experimental attack on these problems. A digital filtering method is proposed for use with reflective macroscopic objects, in order to suppress both the zero-order term and the twin image. Thirdly, for the two-source contouring technique, the following issues have been discussed and thoroughly analysed: the effects of the linear factor, the use of noise reduction filters, different phase unwrapping algorithms, the application of the super-resolution method, and errors in the illumination angle. Practical 3D measurement of a real object, of known geometry, is used as a benchmark for the accuracy improvements achievable via the use of these digital signal processing techniques within the numerical reconstruction stage. The thesis closes by seeking to draw practical conclusions from both the experimental and numerical aspects of the investigation, which it is hoped will be of value to those aiming to use digital holography as a metrology tool.

Dentre dos procedimentos geradores de perfil óptico encontra-se a microscopia holográfica digital. Esta ferramenta interferométrica surgiu da ideia inicial proposta por D. Gabor sobre holografia, a qual permite mediante o registro da interferência de campos ópticos coerentes, guardar e extrair informações de imagens. A microscopia holográfica digital permite a análise de objetos com resoluções transversais semelhantes às obtidas por microscopia óptica, e ainda, possui a vantagem pela natureza da holografia de permitir realizar análises através do acesso a valores quantitativos de fase. Apresentam-se neste trabalho os conceitos básicos da holografia digital e da microscopia holográfica digital, com o objetivo, de introduzir o desenvolvimento de uma metodologia para a implementação da microscopia holográfica digital por reflexão para o controle dimensional de objetos e determinação da rugosidade superficial de amostras de aço. Os hologramas são obtidos mediante uma instalação óptica, que consiste em um interferômetro de Michelson por reflexão com o uso de uma lente objetiva de microscópio e uma câmera CCD sem lente. Para a reconstrução das imagens de contraste de fase são utilizadas técnicas numéricas que capacitam à microscopia holográfica digital para a supressão do termo de ordem zero, controle da resolução de pixel, desmodulação da fase óptica, determinação dos mapas de intensidades e fase, filtragem e compensação de aberrações dos hologramas obtidos. As reconstruções numéricas dos feixes objeto e referência são realizadas utilizando o método de dupla propagação. Foi desenvolvido um algoritmo que apresenta a imagem de contraste de fase com base num critério de distância a partir de um único holograma. Desta forma o programa utilizado permite a realização de medições quantitativas das dimensões dos objetos e da rugosidade superficial de amostras de aço, assim como, a representação em 3D da imagem de fase reconstruída com resultados validados através de um perfilômetro óptico 3D sem contacto modelo CCI-MP. / Among the procedures generating optical profile is the digital holographic microscopy. This interferometric tool arose from the initial idea proposed by D. Gabor on holography, which allows by recording the interference of coherent optical fields, save and extract information from images. Digital holographic microscopy allows the analysis of objects with transversal resolutions similar to those obtained by optical microscopy, and also has the advantage of the nature of holography to allow to perform analyzes through the access to quantitative phase values. This paper presents the basic concepts of digital holography and digital holographic microscopy, with the objective of introducing the development of a methodology for the implementation of digital holographic microscopy by reflection for the dimensional control of objects and determination of surface roughness of samples of steel. The holograms are obtained by means of an optical installation consisting of a Michelson interferometer by reflection using an objective microscope lens and a lensless CCD camera. For the reconstruction of phase contrast images, numerical techniques are used that enable digital holographic microscopy to suppress the zero-order term, control pixel resolution, optical phase demodulation, determination of intensity and phase maps, filtering and compensation of aberrations of the obtained holograms. Numerical reconstructions of the object and reference beams are performed using the double propagation method. An algorithm has been developed that presents the phase contrast image based on a distance criterion from a single hologram. In this way the program used allows the realization of quantitative measurements of the object dimensions and the surface roughness of steel samples, as well as the 3D representation of the reconstructed phase image with results validated through a 3D contactless optical profilometer model CCI- MP.

A Holografia Digital (HD) é uma ferramenta acessível, rápida e eficiente para análise de efeitos mecânicos em materiais fotoelásticos. Esses materiais apresentam a propriedade da dupla refração, ou birrefringência, quando submetidos a esforços externos e, como consequência, os efeitos dos estados de polarização da luz transmitidos através de sua estrutura podem ser utilizados na análise das distribuições de tensões. As técnicas holográficas tradicionais não possibilitam o armazenamento das fases da onda de luz e, portanto, dificultam a análise quantitativa do campo de tensões e deformações nesses materiais. A Holografia Digital permite contornar essa dificuldade por meio de processos que utilizam diretamente as fases da onda de luz armazenadas. A partir de um interferômetro com duas ondas de referências ortogonalmente polarizadas é possível obter hologramas simultâneos que, ao serem subtraídos durante um processo de reconstrução holográfica digital por método da transformada de Fresnel, fornecem diretamente as diferenças de fases. Dessa forma, a HD mostra ser uma alternativa na análise de problemas em diversas áreas, pois possibilita, através de uma única captura, obter informações sobre as propriedades ópticas e mecânicas dos sistemas de interesse. O objetivo desse trabalho foi, além do desenvolvimento instrumental, criar um método para obtenção das distribuições de tensões que surgem nos materiais fotoelásticos e validá-lo por comparação, tanto com um modelo teórico a partir de fundamentos do método de Elementos Finitos associado à Fotoelasticidade quanto com um método experimental de Fotoelasticidade RGB. Com os procedimentos do método proposto, determinou-se as distribuições de tensões em uma amostra fotoelástica acoplada a uma peça metálica para observar a aplicabilidade do método. Essas etapas levaram a resultados que apontam a possibilidade de se utilizar técnicas e métodos holográficos digitais no estudo das propriedades de materiais fotoelásticos. / The Digital Holographic (DH) is an handy, fast and efficient tool to obtain the stresses distributions in Photoelastic materials. These materials present the double refraction phenomenon also named temporary birefringence when subjected to external forces, therefore, the effects in polarization state of the light transmitted through the structure can be used to analyze the stress distribution. Traditional holographic techniques do not allow the storage phase of the light wave dificulting the analyze these of the distributions. Digital Holographic allows to overcome these difficulties by processes that obtain directly of the storage phase of light. The holographic apparatus applied to generate the holograms registered the interference between two orthogonally polarized reference waves and an object wave transmitted through the samples. The resulting phase maps were reconstructed numerically by the Fresnel transform method. Thereby, the DH is an alternative for the study of problems in several fields, because it allows, through a single capture, to obtain information about the optical and mechanical properties of the systems of interest. The objective of this work was, besides instrumental development, create a method in Digital Holography to obtain the stresses distributions in Photoelastic materials and validate it by comparisions between the theorical Finite Element Method associated with Photoelasticity with the experimental method of Photoelasticity RGB. The procedures of the proposed method were used for determine stresses distributions in a Photoelastic sample with a metal part coupled in your superior base to verified its aplicability. Together, these results demonstrate the possibility of using digital holographic techniques and methods for studying mechanical properties of the Photoelastic materials.

Este trabalho teve como objetivo a aplicação do Microscópio Holográfico Digital para análise de amostras biológicas, por meio de imagens de parâmetros físicos e informação quantitativa de uma amostra, gerados através de hologramas digitais, o que não ocorre na holografia clássica. O processamento e análise dos hologramas digitais foi efetuada por um programa escrito por meio do software MatLab, empregando o método de Dupla Propagação. São explicados outros métodos para tratamento de hologramas digitais, presentes no programa. O método de Dupla Propagação foi discutido, destacando suas vantagens frente aos outros métodos. Foi aplicado o método de Volkov para a retirada de ambiguidade de fase. O processo de montagem do Microscópio Holográfico Digital foi descrito, por apresentar modificações em relação ao protótipo inicial adotado. Sete amostras foram analisadas no Microscópio Holográfico Digital, três de calibração e quatro para análise - sangue e solução concentrada de proteína denominada Beta2 Glicoproteína tipo I, ou Beta2-GPI. Para calibração, foram realizados testes de formação de imagem, realizando comparação em quatro microscópios descritos e explicados em funcionamento e princípio envolvidos na formação de imagens, utilizando a mesma amostra; e verificação das dimensões de uma amostra, por meio de medição usando ferramentas disponíveis no programa. Uma amostra de sangue de um indivíduo heterozigoto para Hemoglobina S (anemia falciforme) e uma amostra de sangue de um indivíduo homozigoto para hemoglobina A1 (controle normal) foram empregadas na forma de filmes líquidos secos sobre lâminas de vidro (extensão sanguínea). O uso de fixação foi avaliado com a amostra controle. Foram geradas imagens em duas e três dimensões para as amostras biológicas, reproduzindo as estruturas morfológicas de cada. Para a proteína Beta2-GPI, a análise envolveu somente imagens, sem extração de valores; apesar disso, os resultados mostraram possibilidades de aplicações em estudos futuros. Grandezas físicas foram calculadas para dois dos componentes sanguíneos (Plasma e Eritrócito), mostrando valores próximos daqueles conhecidos anteriormente. Entretanto, alguns valores foram considerados estimativas novas, por não se conhecer, até o momento, nenhum cálculo efetuado anteriormente. A análise comprovou a formação de imagens e a capacidade de mensuração oferecida pelo aparelho. Devido ao parâmetro da fase, foi possível extrair informações em três dimensões. / This work aimed the implementation of the Digital Holographic Microscope for the analysis of biological samples, using physical parameters images and quantitative data from a sample, both generated through digital holograms, which does not occur in Classical holography. Processing and analysis of holograms were performed by a program written using the MatLab software, applying the Double Propagation method. Other methods for the treatment of digital holograms were explained. The Double Propagation method was discussed, highlighting their advantages over other methods. The method of Volkov was applied for removing phase ambiguity. The Digital Holographic Microscope assembly process was described, because of the modifications made to the initial prototype adopted. Seven samples were analyzed in the digital holographic microscope, three of them for calibration and the other to the analysis - blood and a concentrated solution of a protein called type I Beta2 Glycoprotein, or Beta2-GPI. Calibration tests were made by observing and comparing four image microscopes, described and explained in operation and principles involved in the formation of images, using the same testing sample; and checking the dimensions of another sample through measurement, using digital tools available in the program. Hb S heterozygous (Sickle Cell disease) and Hb A1 homozygous (Control) blood samples were prepared in microscope slide glasses. Images were acquired in two and three dimensions for biological samples, reproducing their morphological structures. For Beta2-GPI, the analysis involved only images, and no values were extracted; nevertheless, the results showed potential applications in future studies. Physical quantities were calculated for two blood components (Plasma and Erythrocyte), showing values closer to those previously known. However, some values were considered new estimates, because there is no knowledge of any calculation made previously, until now, using Digital Holographic Microscopy. The analysis proved the formation of images and the measurement capacity offered by the apparatus. Due to the phase parameter, we were able to extract information in three dimensions.

This report is a master thesis, written by an engineering physics and electrical engineering student at Luleå University of Technology.The desires of this project was to remove dislocations from wrapped phase maps using sparse reconstructive methods. Dislocations is an error that can appear in phase maps due to improper filtering or inadequate sampling. Dislocations makes it impossible to correctly unwrap the phasemap.The report contains a mathematical description of a sparse reconstructive method. The sparse reconstructive method is based on KSVDbox which was created by R. Rubinstein and is free for download and use. The KSVDbox is a MATLAB implementation of a dictionary learning algorithm called K-SVD with Orthogonal Matching Pursuit and a sparse reconstructive algorithm. A guide for adapting the toolbox for inpainting is included, with a couple of examples on natural images which supports the suggested adaptation. For experimental purposes a set of simulated wrapped phase maps with and without disloca-tions were created. These simulated phase maps are based on work by P. Picart. The MATLAB implementation that was used to generate these test images can be found in the appendix of this report such that they can easily be generated by anyone who has the interest to do so. Finally the report leads to an outline of five different experiments that was designed to test the KSVDbox for the processing of dislocations. Each one of these experiments uses a different dictionary. These experiments are due to inpainting with, 1. A dictionary based on Discrete Cosine Transform. 2. An adaptive dictionary, where the dictionary learning algorithm has been shown what thearea in the phase map that was damaged by dislocations should look like. 3. An adaptive dictionary, where the dictionary learning algorithm has been allowed to trainon the phase map that with damages. This is done such that areas with dislocations areignored. 4. An adaptive dictionary, where training is done on a separate image that has been designedto contain general phase patterns. 5. An adaptive dictionary, that results from concatenating the dictionaries used in experiment 3 and 4. The first three experiments are complimented with experiments done on a natural image for comparison purposes.The results show that sparse reconstructive methods, when using the scheme used in this work, is unsuitable for processing of dislocations in phase maps. This is most likely because the reconstructive method has difficulties in acquiring a high contrast reconstruction and there is nothing in the algorithm that causes the inpainting from any direction to match with the inpainting from other directions.

Este trabalho teve como objetivo a aplicação do Microscópio Holográfico Digital para análise de amostras biológicas, por meio de imagens de parâmetros físicos e informação quantitativa de uma amostra, gerados através de hologramas digitais, o que não ocorre na holografia clássica. O processamento e análise dos hologramas digitais foi efetuada por um programa escrito por meio do software MatLab, empregando o método de Dupla Propagação. São explicados outros métodos para tratamento de hologramas digitais, presentes no programa. O método de Dupla Propagação foi discutido, destacando suas vantagens frente aos outros métodos. Foi aplicado o método de Volkov para a retirada de ambiguidade de fase. O processo de montagem do Microscópio Holográfico Digital foi descrito, por apresentar modificações em relação ao protótipo inicial adotado. Sete amostras foram analisadas no Microscópio Holográfico Digital, três de calibração e quatro para análise - sangue e solução concentrada de proteína denominada Beta2 Glicoproteína tipo I, ou Beta2-GPI. Para calibração, foram realizados testes de formação de imagem, realizando comparação em quatro microscópios descritos e explicados em funcionamento e princípio envolvidos na formação de imagens, utilizando a mesma amostra; e verificação das dimensões de uma amostra, por meio de medição usando ferramentas disponíveis no programa. Uma amostra de sangue de um indivíduo heterozigoto para Hemoglobina S (anemia falciforme) e uma amostra de sangue de um indivíduo homozigoto para hemoglobina A1 (controle normal) foram empregadas na forma de filmes líquidos secos sobre lâminas de vidro (extensão sanguínea). O uso de fixação foi avaliado com a amostra controle. Foram geradas imagens em duas e três dimensões para as amostras biológicas, reproduzindo as estruturas morfológicas de cada. Para a proteína Beta2-GPI, a análise envolveu somente imagens, sem extração de valores; apesar disso, os resultados mostraram possibilidades de aplicações em estudos futuros. Grandezas físicas foram calculadas para dois dos componentes sanguíneos (Plasma e Eritrócito), mostrando valores próximos daqueles conhecidos anteriormente. Entretanto, alguns valores foram considerados estimativas novas, por não se conhecer, até o momento, nenhum cálculo efetuado anteriormente. A análise comprovou a formação de imagens e a capacidade de mensuração oferecida pelo aparelho. Devido ao parâmetro da fase, foi possível extrair informações em três dimensões. / This work aimed the implementation of the Digital Holographic Microscope for the analysis of biological samples, using physical parameters images and quantitative data from a sample, both generated through digital holograms, which does not occur in Classical holography. Processing and analysis of holograms were performed by a program written using the MatLab software, applying the Double Propagation method. Other methods for the treatment of digital holograms were explained. The Double Propagation method was discussed, highlighting their advantages over other methods. The method of Volkov was applied for removing phase ambiguity. The Digital Holographic Microscope assembly process was described, because of the modifications made to the initial prototype adopted. Seven samples were analyzed in the digital holographic microscope, three of them for calibration and the other to the analysis - blood and a concentrated solution of a protein called type I Beta2 Glycoprotein, or Beta2-GPI. Calibration tests were made by observing and comparing four image microscopes, described and explained in operation and principles involved in the formation of images, using the same testing sample; and checking the dimensions of another sample through measurement, using digital tools available in the program. Hb S heterozygous (Sickle Cell disease) and Hb A1 homozygous (Control) blood samples were prepared in microscope slide glasses. Images were acquired in two and three dimensions for biological samples, reproducing their morphological structures. For Beta2-GPI, the analysis involved only images, and no values were extracted; nevertheless, the results showed potential applications in future studies. Physical quantities were calculated for two blood components (Plasma and Erythrocyte), showing values closer to those previously known. However, some values were considered new estimates, because there is no knowledge of any calculation made previously, until now, using Digital Holographic Microscopy. The analysis proved the formation of images and the measurement capacity offered by the apparatus. Due to the phase parameter, we were able to extract information in three dimensions.

<p>Intracellular motions are important signatures of living tissues, and intracellular dynamics reflect overall cell function and health. Traditional microscopy methods can track 2D cellular motions but do not provide an ensemble evaluation of intracellular activity. Biodynamic imaging (BDI) is a unique 3D imaging technique based on the phase shifts of dynamic light scattering and is highly sensitive to intracellular dynamics in living cells and their changes. This makes BDI a versatile tool to evaluate many different types of samples under various scenarios, and BDI has the potential to improve patient diagnosis and to provide valuable information for health care research. This may include evaluating sample activity, profiling patient chemotherapy response, and studying drug mechanisms. This thesis discusses the theory and modeling of BDI, the construction of BDI systems, sample heterogeneity analysis (TDSI), and the use of BDI to study cytoskeletal drug mechanisms, improve embryo selection and select therapies in pre-clinical trials.</p>

Total Internal Reflection Holographic Microscopy (TIRHM) combines near-field microscopy with digital holography to produce a new form of near-field phase microscopy. Using a prism in TIR as a near-field imager, the presence of microscopic organisms, cell-substrate interfaces, and adhesions, causes relative refractive index (RRI) and frustrated TIR (f-TIR) to modulate the object beam's evanescent wave phase front. Quantitative phase images of test specimens such as Amoeba proteus, Dictyostelium Discoideum and cells such as SKOV-3 ovarian cancer and 3T3 fibroblasts are produced without the need to introduce stains or fluorophores. The angular spectrum method of digital holography to compensate for tilt anamorphism due to the inclined TIR plane is also discussed. The results of this work conclusively demonstrate, for the first time, the integration of near-field microscopy with digital holography. The cellular images presented show a correlation between the physical extent of the Amoeba proteus plasma membrane and the adhesions that are quantitatively profiled by phase cross-sectioning of the holographic images obtained by digital holography. With its ability to quantitatively characterise cellular adhesion and motility, it is anticipated that TIRHM can be a tool for characterizing and combating cancer metastasis, as well as improving our understanding of morphogenesis and embryogenesis itself.

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