Digital holography is a method for recording holograms through the use of an optoelectronic sensor, which serves as a replacement for the use of film [1]. Through the use of coherent light, the microparticles’s characterization can be observed with this method. Yet even using partially coherent light, images of particles can be formed revealing particle shape and size at scales larger than 10 micrometers. For example, ragweed pollen sporeclusters and glass microspheres are investigated here. The holographic images results are compared with conventional optical microscope images for validation.

Digital Holography Microscopy (DHM) is a new 3D measurement technique that exists since Charge Coupled Devices (or CCD cameras) allow to record numerically high resolution images. That opens a new door to the theory of holography discovered in 1949 by Gabor: the door that masked the world of digital hologram processing. A hologram is a usual image but that contains the complex amplitude of the light coded into intensities recorded by the camera. The complex amplitude of the light can be seen as the combination of the energy information (squared amplitude modulus) with the information of the propagation angle of the light (phase of the amplitude) for each point of the image. When the hologram is digital, this dual information associated with a diffractive model of the light propagation permits to numerically investigate back and front planes to the recorded plane of the imaging system. We understand that 3D information can be recorded by a CCD camera and the acquisition rate of this volume information is only limited by the acquisition rate of the unique camera. For each digital hologram, the numerical investigation of front and back regions to the recorded plane is a tool to numerically refocus objects appearing unfocused in the original plane acquired by the CCD. This thesis aims to develop general and robust algorithms that are devoted to automate the analysis process in the 3D space and in time of objects present in a volume studied by a specific imaging system that permits to record holograms. Indeed, the manual processing of a huge amount of holograms is not realistic and has to be automated by software implementing precise algorithms. In this thesis, the imaging system that records holograms is a Mach-Zehnder interferometer working in transmission and studied objects are either of biological nature (crystals, vesicles, cancer cells) or latex particles. We propose and test focus criteria, based on an identical focus metric, for both amplitude and phase objects. These criteria allow the determination of the best focus plane of an object when the numerical investigation is performed. The precision of the best focus plane is lower than the depth of field of the microscope. From this refocus theory, we develop object detection algorithms that build a synthetic image where objects are bright on a dark background. This detection map of objects is the first step to a fully automatic analysis of objects present in one hologram. The combination of the detection algorithm and the focus criteria allow the precise measurement of the 3D position of the objects, and of other relevant characteristics like the object surface in its focus plane, or its convexity or whatever. These extra relevant measures are carried out with a segmentation algorithm adapted to the studied objects of this thesis (opaque objects, and transparent objects in a uniform refractive index environment). The last algorithm investigated in this research work is the data association in time of objects from hologram to hologram in order to extract 3D trajectories by using the predictive Kalman filtering theory. These algorithms are the abstract bricks of two software: DHM Object Detection and Analysis software, and Kalman Tracking software. The first software is designed for both opaque and transparent objects. The term object is not defined by one other characteristic in this work, and as a consequence, the developed algorithms are very general and can be applied on various objects studied in transmission by DHM. The tracking software is adapted to the dynamic applications of the thesis, which are flows of objects. Performance and results are exposed in a specific chapter.

Digital holography (DH) has a variety of applications on measuring the 3D position of different kinds of particles, including the droplets created in drop breakups, seeding particles for flow velocity measurements, characterizations of the behavior of the microorganisms, etc. A particle detection method is required to extract the 3D information encoded in the interference patterns of the holograms, which is desired to be accurate and fast. As the accuracy of the particle detection method improves, the time efficiency of the method decreases. In this study, an optimization process is developed based on an existing method to shorten the processing time. The optimization process includes reducing the complexity of the method and introducing the parallel processing algorithm that can be implemented on cluster machines. The existing particle detection method is separated into several steps and analyzed. The most time consuming step, refining the threshold to separate overlapping particles, is the focus of complexity reduction optimization. A Python code is developed, based on object oriented programming, to implement the optimization. Message Passing Interface (MPI) is applied for parallel processing with a 24-core remote workstation. The optimized Python code is compared with the existing Matlab code in both time consumption and accuracy aspects with synthetic holograms. It is found that the optimization process is able to reduce the time consumption by about four times with an acceptable sacrifice in accuracy. Finally, a DIH system with the optimized method, is applied to characterize different kinds of solid particles. One is noted that the previous studies focus on measuring artificial particles or droplets which are both spherical particles, while most natural solid particles usually have irregular shapes. Equivalent diameter, circularity and aspect ratio are introduced to quantify the dimension and morphology of the irregular shapes. The statistics of the parameters are generated to characterize different kinds of the particles. The accuracy of the characterization of the particles are verified with the observation of the microscopic images of the particles, which can further prove the improvement of the optimized method for particle detection.

A Microscopia Holográfica Digital (MHD) é uma poderosa ferramenta para análise não destrutiva de superfícies e caracterização morfológica e estrutural de materiais. Várias técnicas microscópicas têm sido utilizadas com esses objetivos, mas, em todas elas, a visualização de campo completo não é permitida em uma única aquisição da informação do sistema analisado, além disso, a necessidade de uso de corantes, o processo de varredura, entre outros fatores, dificulta a aquisição das imagens. A Microscopia Holográfica Digital permite contornar essas dificuldades através de um método simples e rápido. Com uma modificação em seu arranjo original, é possível revelar as diferenças na absorção e nos índices de refração experimentados por um campo transmitido por um material anisotrópico. Este método, neste trabalho, foi nomeado como Microscopia Holográfica de Polarização, o qual permite medir as diferenças de fase e de intensidade relacionadas com a birrefringência e com o dicroísmo do material, respectivamente. Dessa forma, A MHD se mostra uma ferramenta promissora na análise de problemas em diversas áreas, pois possibilita, através de uma única captura, obter informações sobre as propriedades ópticas e estruturais dos sistemas de interesse. O objetivo desse trabalho foi, além do desenvolvimento instrumental, analisar variações nas propriedades ópticas e morfológicas de eritrócitos através da quantificação dos índices de refração e perfis topográficos destes quando submetidos aos principais agentes de fixação de amostras. Essas variações são determinantes na escolha do método de análise, já que estes podem alterar significativamente os resultados, assim como a sua interpretação. Também foi descrito um novo método, baseado na técnica holográfica, de análise do dicroísmo linear através de uma varredura executada sobre os padrões de difração das componentes ortogonais do campo transmitido pela amostra. A aplicação da MHD ao estudo de processos dinâmicos também foi realizada, utilizando células cardíacas vivas e proteínas em solução. Somados, estes resultados demonstraram a possibilidade de se utilizar as técnicas holográficas como métodos de estudo amplo de microssistemas biológicos. / The Digital Holographic Microscopy (DHM) is a powerful tool for nondestructive analysis of surfaces and for morphological and structural characterization of materials. Several microscopic techniques have been used with these purposes, but in all cases, the complete field reconstruction is not allowed in a single acquisition of information of the analyzed system, moreover, the requirements of staining and scanning procedures, among other factors, difficult the image acquisition. Digital Holographic Microscopy allows to overcome these difficulties through a simple and quick method. With a modification in its original setup, is possible to reveal the differences in absorption and refractive index experienced by a wavefield transmitted by an anisotropic material. This method, in this thesis, is named Polarization Holographic Microscopy, which allows to measure differences in phase and intensity related to the birefringence and the dichroism of the material, respectively. Thereby, the DHM is shown as a promising tool for the study of problems in several fields, because it allows, through a single capture, to obtain information related to optical and morphological properties of the systems of interest. The objective of this work was, besides instrumental development, to evaluate changes in optical and morphological properties of erythrocytes through quantification of their refractive index and topographic profiles when submitted to the main fixating agents. These changes are crucial once the method of analysis is chosen, as they can significantly alter the results, as well as their interpretation. A new method was also described based on the holographic technique, for analysis of linear dichroism, through scans performed on the diffraction patterns of the orthogonal components of the field transmitted by the sample. The application of the DHM to the study of dynamic processes was also performed, using live cardiac cells and proteins in solution. Together, these results demonstrate the possibility of using holographic techniques such as extensive methods for studying biological microsystems.

When a particle is illuminated by a beam of light it will scatter and redistribute the light in all directions. How it scatters depends on the size, shape and refractive index of the particle. Additionally, it depends on the wavelength and polarization of the illuminating beam. The direction and distance to the observer relative the particle also needs to be considered.  A digital holographic imaging system is used to collect parts of the scattered light from micrometer-sized particles. By utilizing digital holography a three-dimensional reconstruction of the imaged scene is possible. Traditionally, particles are localized based on the intensity in the holographic reconstructions. In this licentiate thesis, the phase response of the scattered light is investigated and utilized. An alternative method for locating spherical particles is presented. The method locate particles based on a simple feature of a propagating wave, namely the fact that the wavefront curvature changes from converging to diverging at the axial location of the particle. The wavefront curvature is estimated using two different methods. The first method estimates the lateral phase-gradients using a finite-difference method. The second method uses a three-dimensional parametric model based on a Chebyshev polynomial expansion. The methods are demonstrated using both simulations and experimental measurements. The simulations are based on the Lorenz-Mie scattering theory for spherical particles and are combined with an imaging system model. Experiments are performed using an off-axis polarization sensitive digital holographic system with a coherent Nd:YAG laser. Measurements of stationary particles are made to validate and evaluate the proposed method. It is found that these methods estimate the true axial position and does not have the offset that is associated with intensity-based methods. Additionally, it is possible to exclude noise that shows up as false particles since noise does not have the same phase response as a real particle. The second method, that uses a parametric model, also improves the standard deviation in the positioning.

A Microscopia Holográfica Digital (MHD) é uma poderosa ferramenta para análise não destrutiva de superfícies e caracterização morfológica e estrutural de materiais. Várias técnicas microscópicas têm sido utilizadas com esses objetivos, mas, em todas elas, a visualização de campo completo não é permitida em uma única aquisição da informação do sistema analisado, além disso, a necessidade de uso de corantes, o processo de varredura, entre outros fatores, dificulta a aquisição das imagens. A Microscopia Holográfica Digital permite contornar essas dificuldades através de um método simples e rápido. Com uma modificação em seu arranjo original, é possível revelar as diferenças na absorção e nos índices de refração experimentados por um campo transmitido por um material anisotrópico. Este método, neste trabalho, foi nomeado como Microscopia Holográfica de Polarização, o qual permite medir as diferenças de fase e de intensidade relacionadas com a birrefringência e com o dicroísmo do material, respectivamente. Dessa forma, A MHD se mostra uma ferramenta promissora na análise de problemas em diversas áreas, pois possibilita, através de uma única captura, obter informações sobre as propriedades ópticas e estruturais dos sistemas de interesse. O objetivo desse trabalho foi, além do desenvolvimento instrumental, analisar variações nas propriedades ópticas e morfológicas de eritrócitos através da quantificação dos índices de refração e perfis topográficos destes quando submetidos aos principais agentes de fixação de amostras. Essas variações são determinantes na escolha do método de análise, já que estes podem alterar significativamente os resultados, assim como a sua interpretação. Também foi descrito um novo método, baseado na técnica holográfica, de análise do dicroísmo linear através de uma varredura executada sobre os padrões de difração das componentes ortogonais do campo transmitido pela amostra. A aplicação da MHD ao estudo de processos dinâmicos também foi realizada, utilizando células cardíacas vivas e proteínas em solução. Somados, estes resultados demonstraram a possibilidade de se utilizar as técnicas holográficas como métodos de estudo amplo de microssistemas biológicos. / The Digital Holographic Microscopy (DHM) is a powerful tool for nondestructive analysis of surfaces and for morphological and structural characterization of materials. Several microscopic techniques have been used with these purposes, but in all cases, the complete field reconstruction is not allowed in a single acquisition of information of the analyzed system, moreover, the requirements of staining and scanning procedures, among other factors, difficult the image acquisition. Digital Holographic Microscopy allows to overcome these difficulties through a simple and quick method. With a modification in its original setup, is possible to reveal the differences in absorption and refractive index experienced by a wavefield transmitted by an anisotropic material. This method, in this thesis, is named Polarization Holographic Microscopy, which allows to measure differences in phase and intensity related to the birefringence and the dichroism of the material, respectively. Thereby, the DHM is shown as a promising tool for the study of problems in several fields, because it allows, through a single capture, to obtain information related to optical and morphological properties of the systems of interest. The objective of this work was, besides instrumental development, to evaluate changes in optical and morphological properties of erythrocytes through quantification of their refractive index and topographic profiles when submitted to the main fixating agents. These changes are crucial once the method of analysis is chosen, as they can significantly alter the results, as well as their interpretation. A new method was also described based on the holographic technique, for analysis of linear dichroism, through scans performed on the diffraction patterns of the orthogonal components of the field transmitted by the sample. The application of the DHM to the study of dynamic processes was also performed, using live cardiac cells and proteins in solution. Together, these results demonstrate the possibility of using holographic techniques such as extensive methods for studying biological microsystems.

Cette thèse traite de l'application de l'holographie numérique dans l'axe à l'étude d'une dynamique tourbillonnaire dans un champ de houle. Lors du passage de la houle au dessus d'une structure immergée, des tourbillons se forment à ses extrémités. Ces tourbillons ont un impact fort sur la dynamique proche des structures et peuvent engendrer des problèmes d'affouillement ou de vieillissement prématuré. On s'intéresse donc à la dynamique tridimensionnelle de ces tourbillons qui sont rapidement l'objet de déformations menant à leur éclatement. Pour étudier ce mécanisme, le problème est modélisé par une géométrie simple, une plaque soumise à une houle monochromatique. Les expériences sont menées dans un canal à houle de 10 m de long et 30 cm de large. Pour mesurer la dynamique 3D on utilise une technique innovante, l'holographie numérique qui permet une mesure 3D3C au moyen d'une diode laser et d'une seule caméra. Ses principales limitations résident dans la dimension de la section du volume de mesure, qui est de l'ordre de la surface du capteur CCD, ainsi que du nombre de particules présentes dans le champ. Toutefois, l'holographie permet une résolution de l'ordre de la taille d'un pixel dans le plan du capteur CCD et de 3 à 5 fois le diamètre de la particule suivie dans la direction orthogonale au capteur. Ces mesures permettent de suivre individuellement plusieurs centaines de particules dans le champ et d'en mesurer les vitesses. Enfin des mesures complémentaires en stéréo-piv confirment les résultats obtenues par holographie et permettent l'étude du confinement du tourbillon sous l'action combinée de la surface libre et de la plaque. / This thesis deals with the use of digital in-line holography to the study of a vortex dynamics under water waves. As waves propagate above an immersed structure, vortices are formed at its edges. Those vortices have a strong impact on the flow dynamics in the vicinity of structures and can cause scouring or damages. Thus we are interested in the three-dimensional dynamics of those vortices which are quickly distorted, leading to their breakup. To study this dynamics, the physical problem is modelled by a basic geometry, a thin plate is set under monochromatic waves. Experiments are conduct within a wave flume of dimensions 10 m long and 30 cm width. To measure the 3D flow the use of an innovative technique, the digital holography which allow a 3D3C measure with only one camera and a laser diode. Its main limitations are the size of the cross-section of the sample volume and the number of particles allowed in it. However, digital holography can localize particles with a pixel sized resolution within the plans parallel to the CCD sensor and a depth resolution in the order of 3 to 5 times the particles diameter. Those measurements enable to follow the path of each particle inside the sample volume. Finally, acquisition by stereo particle image velocimetry confirms the velocities measured by holography and are used to study the interaction between the vortex and the combined action of free surface and the plate.

The digital in-line holographic configuration is motivated by the goal of developing a portable, cost effective sensor system for pre-screening patient blood samples. The theory of holography is explained from the foundational concepts in scalar diffraction theory all the way through to the implementation of reconstruction algorithms. Methods for the enhancement of holographic reconstructions are described. The algorithms that perform an automated count of the reconstructed objects are described and demonstrated. Simulated and experimental results are provided. Together, the lens-free holographic microscopy of micro-sized particles along with the application of image processing techniques for the automated detection and counting of objects of interest, provide a component towards realising a sensor system that can be used for pre-screening patient blood samples. / Dissertation (MSc)--University of Pretoria, 2017. / CSIR / Computer Science / MSc / Unrestricted

<p>Biodynamic imaging (BDI) is a high-content optical imaging technology based on Fourier-domain digital holography and Doppler spectroscopy of intracellular dynamics. There are three main functions of the BDI technique, which are optical coherence imaging (OCI), motility contrast imaging (MCI) and tissue dynamics spectroscopy (TDS). OCI is related to <em>en face</em> optical coherence tomography (OCT) using partially coherent speckle generated by broad-area illumination with coherence detection through digital holography. MCI provides noninvasive functional imaging by treating intracellular motility as an endogenous dynamic imaging contrast agent. TDS produces broad-band Doppler fluctuation power spectra that contain the ensemble of all intracellular motions by collecting and extracting depth-resolved quasi-elastic dynamic light scattering from inside multicellular living tissue. This thesis presents the development and applications of BDI systems. Doppler spectral clustering analysis is demonstrated when comparing fresh canine lymphoma biopsies and their corresponding flash-Frozen samples. Doppler spectral phenotyping analysis is used to identify a non-predictive phenotype of TDS that shows a systemic red-shift of frequencies. Doppler spectral shift analysis is used to monitor bacterial infection of living tissue. </p>

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