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.

A novel optical calorimetry approach is proposed for the dosimetry of therapeutic radiation, based on the optical technique of Digital Holographic Interferometry (DHI). This detector determines the radiation absorbed dose to water by measurement of the refractive index variations arising from radiation induced temperature increases. The output consists of a time series of high resolution, two dimensional images of the spatial distribution of the projected dose map across the water sample. This absorbed dose to water is measured directly, independently of radiation type, dose rate and energy, and without perturbation of the beam. These are key features which make DHI a promising technique for radiation dosimetry. A prototype DHI detector was developed, with the aim of providing proof-of-principle of the approach. The detector consists of an optical laser interferometer based on a lensless Fourier transform digital holography (LFTDH) system, and the associated mathematical reconstruction of the absorbed dose. The conceptual basis was introduced, and a full framework was established for the measurement and analysis of the results. Methods were developed for mathematical correction of the distortions introduced by heat di usion within the system. Pilot studies of the dosimetry of a high dose rate Ir-192 brachytherapy source and a small eld proton beam were conducted in order to investigate the dosimetric potential of the technique. Results were validated against independent models of the expected radiation dose distributions. Initial measurements of absorbed dose demonstrated the ability of the DHI detector to resolve the minuscule temperature changes produced by radiation in water to within experimental uncertainty. Spatial resolution of approximately 0.03 mm/pixel was achieved, and the dose distribution around the brachytherapy source was accurately measured for short irradiation times, to within the experimental uncertainty. The experimental noise for the prototype detector was relatively large and combined with the occurrence of heat di usion, means that the method is predominantly suitable for high dose rate applications. The initial proof-of-principle results con rm that DHI dosimetry is a promising technique, with a range of potential bene ts. Further development of the technique is warranted, to improve on the limitations of the current prototype. A comprehensive analysis of the system was conducted to determine key requirements for future development of the DHI detector to be a useful contribution to the dosimetric toolbox of a range of current and emerging applications. The sources of measurement uncertainty are considered, and methods suggested to mitigate these. Improvement of the signal-to-noise ratio, and further development of the heat transport corrections for high dose gradient regions are key areas of focus highlighted for future development.

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.

Reflected-light digital holographic microscope developed at IPE FME BUT uses off-axis holography principle and low spatial and temporal coherence illumination. Microscope allows reconstruction of the image amplitude and the image phase, which can be handled in real time. The only limiting factors are imaging speed of the detector and computer performance when processing holograms. Reconstruction of image phase and amplitude allows high-resolution profilometric measurements in the vertical axis direction. This thesis deals with the automatization of profilometric measurement method proposed in [2]. Proposed method uses the combination of the image phase and the image amplitude for the measurement of specimens with surface structure the vertical size of which cause the uncertainty of the image phase by a factor of 2pí. Futher the thesis deals with the construction design of the illumination system of the microscope and its realization together with experimental verification of functionality of proposed method automatization.

The diploma thesis deals with theory of imaging in a transmitted-light digital holographic microscope using partially coherent illumination. The influence of spatial and temporal coherence state on optical sectioning property is solved. The coherent transfer function is calculated. From this function imaging characteristics for a two-dimensional scattering object are derived depending on its defocus. Two different designs of microscopes developed in the Laboratory of optical microscopy in IPE FME BUT are considered.