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.

For the proper function of the Fluorescence olographic microscope, it is necessary to adjust all the optical components of the microscope. Furthermore, the precise adjustment is the very critical condition for proper imaging of the Coherence-controlled holographic microscope. Therefore, it is necessary to create a sight collimator for these microscopes for their adjustment. The fluorescence holographic microscope is based on an interference and holographic principles, whose history is mentioned in the theoretical part of the thesis. The existing state of the art of laser sight collimators and their use in practice is also mentioned. The optical and mechanical design of the laser sight collimator and its realization are described in the next part of the thesis. The software for detecting the black sight cross was created for the use of the laser sight collimator in practice. The software is necessary to evaluate the correctness of the alignment of the adjusted microscope. The descriptions of the adjustment procedures for the laser sight collimator and for the Fluorescence holographic microscope are mentioned in the last part of the thesis. These procedures are necessary for proper manipulation and use with the proposed laser sight collimator.

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.

Microfluidics is a very recent branch of science and technology. The development and the success, it has had in the last 15 years, is mainly due to the concept of lab-on-a-chip. Those miniaturized devices, integrating one or more laboratory functions, have aroused great interest among several research areas as physics, chemistry, biology and bioengineering. When a fluid is confined in a micro or nano scale structure, its behaviour is strongly affected by its interactions with the surrounding surfaces. In this context, the theme of fluid/solid slippage has been widely studied both theoretically and experimentally. Innovative technologies to enhance the surface slippage by specifically designing the solid interfaces have reportedly demonstrated to be an effective way to reduce the fluid/solid friction. To this end, superhydrophobic surfaces have increasingly attracted the interest of the scientific and technological community thanks to the large wall-slippage they present for liquid water. Though their behaviour has been extensively investigated through several theoretical and numerical methods, the experimental approaches are still indispensable to test and understand the properties of these surfaces. However, the lack of a general predicting model is also due to the fact that no one of the several existing experimental techniques has shown up as a very reliable one. Indeed, the reported measurements of slippage still depends on the specific adopted method, thwarting attempts to corroborate the proposed theoretical and numerical schemes. Therefore, it is evident that a more sensitive and effective experimental technique is still missing. This thesis began and developed inside the wider project of setting up an innovative technique to investigate the fluid-solid slippage on superhydrophobic surfaces by means of optical tweezers. Even though this project is still going on, this work reports the steps performed along the long way towards this main goal and it consists of a collection of several researches involving different scientific fields as optics, microscopy, surface science, microhydrodynamics, microfluidics and microfabrication. The researches presented in this work can be separated in two main categories: i) holographic micromanipulation and microscopy, ii) superhydrophobicity.

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.

La microfluidique est une branche récente de la science et de la technologie. Ces quinze dernières années, son développement et son succès ont été principalement dus au concept de labo sur puce (lab-on-a-chip). Ces dispositifs miniaturisés, qu'intègrent plusieurs fonctions de laboratoire, ont été d'un grand intérêt dans différents domaines comme la Physique, la Chimique et la Bio-ingénierie. Lorsque un fluide est confiné dans une structure micro ounanométrique, son comportement est fortement influencé par les interactions avec les surfaces qui l'entourent. Dans ce contexte, la problématique du glissement du fluide sur le solide a été largement étudiée soit théoriquement, soit expérimentalement. Des nouvelles technologies, destinées à augmenter le glissement se sont déjà avérées être une méthode très efficace pour la réduction du frottement fluide/solide. Dans ce contexte, les surfaces superhydrophobes ont sucité l'intérêt de la communauté scientifique et technologique grâce au grand glissement à la paroi qui caractérise ces surfaces. Même si le comportement de ces surfaces a été largement étudié par différentes méthodologies théoriques et numériques, l'approche expérimentale est encore indispensable pour tester et comprendre les propriétés de ces surfaces. Cette thèse a commencé et s'est développée dans un projet plus large qui porte sur la mise en place d'une nouvelle technique pour l'analyse du glissement liquide/solide sur les surfaces superhydrophobes à travers les pièges optiques. Ce manuscrit développe les étapes du projet de recherche qui concernent différents domaines scientifiques comme l'optique, la microscopie, la science des surfaces, la microhydrodynamique, la microfludique et la microfabrication. Les recherches présentées dans cette thèse sont divisées par deux catégories: i) micromanipulation et microscopie holographique, ii) superhydrophobie. / Microfluidics is a very recent branch of science and technology. The development and the success, it has had in the last 15 years, is mainly due to the concept of lab-on-a-chip. Those miniaturized devices, integrating one or more laboratory functions, have aroused great interest among several research areas as physics, chemistry, biology and bioengineering. When a fluid is confined in a micro or nano scale structure, its behaviour is strongly affected by its interactions with the surrounding surfaces. In this context, the theme of fluid/solid slippage has been widely studied both theoretically and experimentally. Innovative technologies to enhance the surface slippage by specifically designing the solid interfaces have reportedly demonstrated to be an effective way to reduce the fluid/solid friction. To this end, superhydrophobic surfaces have increasingly attracted the interest of the scientific and technological community thanks to the large wall-slippage they present for liquid water. Though their behaviour has been extensively investigated through several theoretical and numerical methods, the experimental approaches are still indispensable to test and understand the properties of these surfaces. However, the lack of a general predicting model is also due to the fact that no one of the several existing experimental techniques has shown up as a very reliable one. Indeed, the reported measurements of slippage still depends on the specific adopted method, thwarting attempts to corroborate the proposed theoretical and numerical schemes. Therefore, it is evident that a more sensitive and effective experimental technique is still missing. This thesis began and developed inside the wider project of setting up an innovative technique to investigate the fluid-solid slippage on superhydrophobic surfaces by means of optical tweezers. Even though this project is still going on, this work reports the steps performed along the long way towards this main goal and it consists of a collection of several researches involving different scientific fields as optics, microscopy, surface science, microhydrodynamics, microfluidics and microfabrication. The researches presented in this work can be separated in two main categories: i) holographic micromanipulation and microscopy, ii) superhydrophobicity.

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.

Diplomová práca sa zaoberá kvantitatívnym fázovým zobrazovaním objektov umiestnených za rozptyľujúcim prostredím v koherenciou riadenom holografickom mikroskope. Tento mikroskop umožnuje zobrazovať s úplne nekoherentným osvetlením vzorky, čo vyvoláva efekt koherenčnej brány. Koherenčná brána je veľmi dôležitá vlastnosť zobrazovacieho systému umožňujúca separáciu balistických a rozptýlených fotónov, jej dôkladnému vysvetleniu je venovaná značná časť práce. Ďalej sú prezentované základy teórie zobrazenia v koherenciou riadenom holografickom mikroskope. Tie sú využité v závere práce pri interpretácii experimentálnych výsledkov. Cieľom práce je navrhnúť metódu pre pozorovanie fázových objektov v rozptyľujúcich prostrediach a experimentálne túto metódu overiť. Na základe analytických výsledkov a predchádzajúceho výskumu je navrhnutá nová metóda, ktorá je ďalej overovaná pomocou rôzne komplexných vzoriek. Je založená na zázname viacerých obrazov s rôznym posunutím referenčného poľa. Každý posun korešponduje so zobrazovaním pomocou inej skupiny fotónov. Je možné vytvoriť syntetický obraz so zlepšenou kvalitou sčítaním jednotlivých obrazov získaných z interferencie balistických alebo rozptýlených fotónov. Experimenty s rôzne komplexnými vzorkami poskytujú náhľad na obmedzenia prezentovanej metódy.

This thesis deals with the problem of practical application of FDTD technique for simulation of image formation in coherence controlled holographic microscope. Various ways for obtaining scattering matrices are explored in detail and the optimal technique based on a rigorous calculation of the far field is proposed. The scattering matrix, containing information about the observed sample, is subsequently used in analytic calculation of holographic signal; two levels of approximation of pupil function are also evaluated. The results are compared with a traditional approach based on Rytov approximation resulting in specification of the parameter domain where the approximation is applicable. Based on the simulations of the microscope, the dependence of axial resolution on apertures of the objective and the condenser and sensitivity of the signal to changes of refractive index of the sample is also studied.