In this work, digital holography (DH) is extensively utilized to characterize microparticles. Here, “characterization” refers to the determination of a particle’s shape, size, and, in some cases, its surface structure. A variety of microparticles, such as environmental dust, pollen, volcanic ash, clay, and biological samples, are thoroughly analyzed. In this technique, the microscopically fine interference pattern generated by the coherent superposition of an object and a reference wave fields is digitally recorded using an optoelectronic sensor, in the form of a hologram, and the desired particle property is then computationally extracted by performing a numerical reconstruction to form an image of the particle. The objective of this work is to explore, develop, and demonstrate the feasibility of different experimental arrangements to reconstruct the image of various arbitrary-shaped particles. Both forward- and backward-scattering experimental arrangements are constructed and calibrated to quantify the size of several micron-sized particles. The performance and implications of the technique are validated using the National Institute of Standards and Technology (NIST)-traceable borosilicate glass microspheres of various diameters and a Thorlabs resolution plate. After successful validation and calibration of the system, the resolution limit of the experimental setup is estimated, which is ~10 microns. Particles smaller than 10 microns in size could not be imaged well enough to ensure that what appeared like a single particle was not in fact a cluster. The forward- and backward-scattering holograms of different samples are recorded simultaneously and images of the particles are then computationally reconstructed from these recorded holograms. Our results show that the forward- and backward-scattering images yield different information on the particle surface structure and edge roughness, and thus, reveal more information about a particle profile. This suggests that the two image perspectives reveal aspects of the particle structure not available from a more commonly used forward-scattering based image alone. The results of this work could be supportive to insight more on the particles’ morphology and subsequently important for the advancement of contactree particle characterization technique.

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.

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.

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.

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.

Digital holographic microscopy is an interferometric imaging technique, the principle of which is the off-axis image plane holography. The principle of this technique enables to reconstruct both the image intensity and the image phase from the output interferencesignal. The reconstruction can be carried out on the basis of a single image plane hologram. This leads to the possibility of a realtime image reconstruction. The speed of the reconstruction depends on the detection and the computing process. The aim of this diploma thesis is to develop user software for the control of the detection camera and for the image plane hologram reconstruction. The effort was to achieve the highest number of image reconstructions per time unit, with the maximum utilization of the data transfer between the camera and the computer.The next aim of this thesis is the stabilization of the optical table position. The method of stabilization is based on the image phase information, which is used for the control loop feedback between reconstructed image phase and the piezoelectric actuator placed inside of the optical table. Experimental results, which prove the functionality of the stabilization, are presented.

Holography has the ability to render three-dimensional information of a recorded scene by capturing both the amplitude and phase of light incident on the recording medium. The application of digital camera technology and high-speed computing means digital holograms can be analysed numerically and novel applications can be found for this technology. This thesis explores the potential for both inline and off-axis digital holographic microscopy to study the three-dimensional swimming behaviour of bacteria. A high-magnification (225x) digital holographic microscope was designed and constructed with the ability to switch easily between inline and off-axis imaging modalities. Hardware aspects, in particular the illumination source, the choice of camera and data transfer rates, were considered. Novel strategies for off-axis holography combining dark field microscopy were designed and implemented. The localisation accuracy of the inline imaging modality was assessed by studying samples of polystyrene microspheres. The microscope is sensitive to stage drift on the order of angstroms per second and can successfully localise microspheres in dilute suspensions at least 100μm from the objective specimen plane. As a simple test of the capabilities of the microscope, the diffusion coefficient of a 0.5μm microsphere was found to be isotropic and consistent with the theoretical value. Amplitude and phase image reconstructions from the off-axis modality are demonstrated. High-magnification dark field off-axis holographic microscopy is shown to be superior to inline microscopy in localising 100nm gold nanoparticles. An artifact from our method of dark-field imaging, however, restricts the depth range to 15μm. A lower-magnification (45x) configuration of the microscope was used to study the 3D swimming behaviour of wild type Escherichia coli as a qualitative demonstration of the potential for this instrument in microbiological applications.

Moving towards label-free technologies is essential for many clinical and research applications. Raman spectroscopy is a powerful tool in the field of biomedicine for label-free cell characterisation and disease diagnosis, owing to its high chemical specificity. However, Raman scattering is a relatively weak process and can require long acquisition times, thus hampering its integration to clinical technologies. Multimodal analysis is currently pushing the boundaries in biomedicine, obtaining more information than would be possible using a single mode and overcoming any limitations specific to a single technique. Digital holographic microscopy (DHM) is a rapid and label-free quantitative phase imaging modality, providing complementary information to Raman spectroscopy, and is thus an ideal candidate for combination in a multimodal system. Firstly, this thesis explores the use of wavelength modulated Raman spectroscopy (WMRS), for the classification of immune cell subsets. Following this a multimodal approach, combining Raman spectroscopy and DHM, is demonstrated, where each technique is considered individually and in combination. The complementary modalities provide a wealth of information (both chemical and morphological) for cell characterisation, which is a step towards achieving a label-free technology for the identification of human immune cells. The suitability of WMRS to discriminate between closely related neuronal cell types is also explored. Furthermore optical spectroscopic techniques are useful for the analysis of food and beverages. The use of Raman and fluorescence spectroscopy to successfully discriminate between various whisky and extra-virgin olive oil brands is demonstrated, which may aid the detection of counterfeit or adulterated samples. The use of a compact Raman device is utilised, demonstrating the potential for in-field analysis. Finally, monodisperse and highly spherical nanoparticles are synthesised. A short study demonstrates the potential for these nanoparticles to benefit the techniques of surface enhanced Raman spectroscopy and optical trapping, by way of minimising variability.

The main objective of this thesis is the development of new processing techniques for digital holograms. The present work is part of the HoloFlow project that intends to integrate the DHM technology for the monitoring of water quality. Different tools for an automated analysis of digital holograms have been developed to detect, refocus and classify particles in continuous fluid flows. A detailed study of the refocusing criterion permits to determine its dependencies and to quantify its robustness. An automated detection procedure has been developed to determine automatically the 3D positions of organisms flowing in the experiment volume. Two detection techniques are proposed: a usual method based on a global threshold and a new robust and generic method based on propagation matrices, allowing to considerably increase the amount of detected organisms (up to 95 %) and the reliability of the detection. To handle the case of aggregates of particles commonly encountered when working with large concentrations, a new separation procedure, based on a complete analysis of the evolution of the focus planes, has been proposed. This method allows the separation aggregates up to an overlapping area of around 80 %. These processing tools have been used to classify organisms where the use of the full interferometric information of species enables high classifier performances to be reached (higher than 93 %). / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Práce se zabývá interpretací kvantitativního fázového zobrazení pomocí techniky koherencí řízené holografické mikroskopie. Vzhledem k tomu, že tato technika generuje velké množství kvantitativních fázových obrazů o nezanedbatelné velikosti, manuální analýza by byla časově náročná a neefektivní Za účelem urychlení analýzy obrazů získaných pomocí koherencí řízené holografické mikroskopie je v této práci navržena metodika automatizované interpretace kvantitativních fázových obrazů pomocí strojového učení s učitelem. Kvantitativní fázové obrazy umožňují extrakci parametrů charakterizujících distribuci suché hmoty v buňce a poskytují tak cennou informaci o buněčném chování. Cílem této práce je navrhnout metodologii pro automatizovanou klasifikaci buněk při využití této kvantitativní informace jak ze statických, tak z časosběrných kvantitativních fázových obrazů. Navržená metodika byla testována v experimentech s živými buňkami, jimiž byla vyhodnocena výkonnost klasifikace a významnost parametrů získaných z kvantitativních fázových obrazů.