Bio-pharmaceutical industry is a vast growing market and recent recommendations of the Food and Drug Administration have put a large emphasis on the characterization of biological processes and models. As a consequence, there is a high incentive on developing modern sensors in order to more accurately monitor and control processes. In that way, Digital Holographic Microscopy (DHM) presents unique features thanks to the refocusing and quantitative phase contrast imaging capabilities. In this thesis we investigate the usage of DHM to monitor yeast cultures that are often used in both the bio-pharmaceutical and bread industries and lay the basis of a methodological framework for the study of in-line cell cultures in the context of process control. We begin with a description of Digital Holography and the microscopy setup used in the thesis as well as a detailed explanation of the image processing required to extract the holographic data and its implementation on GPU with some speed execution figures given for three popular programming paradigms. We then describe the flow setup used and infer the limitations on the dynamic range of the technique due to both Poisson statistics and overlapping phenomena. Finally, we describe an algorithm that extracts the cells position, count and morphological information such as the size, aspect ratio, circularity and refraction index. Some experimental results are presented for yeasts before drawing a general overview of the technology and its dependencies. We further end with some conclusions concerning the technology and a brief comparison with existing competitors. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

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.<p>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. <p>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. <p> / Doctorat en sciences appliquées / info:eu-repo/semantics/nonPublished

Nous présentons une nouvelle technique de microscopie stochastique basée sur un montage d'Holographie Digitale pour l'imagerie des distributions d'intensité optique. Nous montrons comment cette technique de champ lointain peut être adaptée afin d'obtenir des images de superrésolution ainsi que de champ proche. En pratique, nous imageons des nanoparticules métalliques en mouvement Brownien dans un liquide, que nous localisons ensuite dans le but de contourner la limite de diffraction. Le mouvement aléatoire des particules nous permet une exploration complète de l'échantillon. Au-delà de la simple localisation, ces marqueurs métalliques agissent comme des sondes locales du champ électromagnétique, pouvant notamment diffuser la lumière confinée vers le champ lointain. Les possibilités de cette nouvelle technique sont illustrées à travers l'imagerie de l'intensité optique d'une onde évanescente et d'une onde propagative. Grâce à des méthodes de calcul très performantes, nous sommes capables de localiser des centaines de particules par minute, avec une précision de l'ordre de 3×3×10 nm3 pour des particules immobiles. En plus de l'imagerie des distributions de champ optique, nous présentons une application combinant nos mesures superrésolues et des mesures d'électrochimie pour l'étude des processus d'oxydation de nanoparticules d'argent à proximité d'une électrode. Nos résultats ouvrent la voie à une nouvelle technique d'imagerie superrésolue, particulièrement bien adaptée à la caractérisation optique dans des milieux liquides (comme des systèmes microfluidiques), qui étaient jusqu'à présent inaccessibles par microscopie électronique ou par des microscopies à sonde locale. / In this thesis work, we present a novel stochastic microscopy technique based on Digital Holography for the 3D mapping of optical intensity distributions. We show that this far-field, wide-field, 3D microscopy can be turned into both a superresolution and a near-field imaging technique. To do so, we use metallic nanoparticles undergoing Brownian motion as stochastic local field probes that we localize in three-dimensions in order to overcome the diffraction limit. The random motion of the particles allows for a complete exploration of the sample. Beyond simple localization, the gold markers can actually be envisaged as extremely local electromagnetic field probes, able to scatter light into the far-field. The technique we propose here is therefore a combination of the concepts of superlocalization and NSOM microscopies. The possibilities of the technique are illustrated through the 3D optical mapping of an evanescent and a propagative wave. Fast computation methods allow us to localize hundreds of particles per minute with accuracies as good as 3×3×10 nm3 for immobilized particles. In addition to optical intensity mapping, we show a particular application in electrochemistry, by coupling our high resolution images with electrochemical oxidation measurements on silver nanoparticles in solution at the vicinity of an electrode. Our results pave the way for a new subwavelength imaging technique, well adapted to optical characterization in water-based systems (such as in emerging microfluidics studies), which are mostly inaccessible to electron microscopy or local probe microscopies.

Deformable mirrors are the standard adaptive optical elements for aberration correction in confocal microscopy. Their usage leads to increased contrast and resolution. However, these improvements are achieved at the cost of bulky optical setups. Since spherical aberrations are the dominating aberrations in confocal microscopy, it is not required to employ all degrees of freedom commonly offered by deformable mirrors. In this contribution, we present an alternative approach for aberration correction in confocal microscopy based on a novel adaptive lens with two degrees of freedom. These lenses enable both axial scanning and aberration correction, keeping the setup simple and compact. Using digital holography, we characterize the tuning range of the focal length and the spherical aberration correction ability of the adaptive lens. The operation at fixed trajectories in terms of focal length and spherical aberrations is demonstrated and investigated in terms of reproducibility. First results indicate that such adaptive lenses are a promising approach towards high-resolution, high-speed three-dimensional microscopy.

In this contribution we use wavefront shaping approaches for image correlation based flow-field measurements for the first time. Aberrations introduced by a single phase boundary in the detection beam path were explored. Variations of the optical path-length result in strong errors in position allocation and thus to an enhancement of the measurement uncertainty of the velocity. Our results show that the usage of wavefront shaping enables to reduce these errors and to strongly improve the quality of image correlation based flow-field measurements. First experimental and simulated results underline the importance of these approaches.

We propose the usage of wavefront shaping approaches for image correlation based flow-field measurements. Aberrations introduced by a single phase boundary in the detection beam path were explored. Variations of the optical path-length result in strong errors in position allocation and thus to an enhancement of the measurement uncertainty of the velocity. Our results show that the usage of wavefront shaping enables to reduce these errors and to strongly improve the quality of image correlation based flow-field measurements. First experimental and simulated results underline the importance of these approaches

Variations of the optical detection path-length in image correlation based flow-field measurements result in strong errors in position allocation and thus lead to a strong enhancement of the measurement uncertainty of the velocity. In this contribution we use digital holography to measure the wavefront distortion induced by fluctuating phase boundary, employing spatially extended guide stars. The measured phase information is used to correct the influence of the phase boundary in the detection path employing a spatial light modulator. We analyze the potential of guide stars that are reflected by the phase boundary, i.e. the Fresnel reflex, and transmitted. Our results show, that the usage of wavefront shaping enables to strongly reduce the measurement uncertainty and to strongly improve the quality of image correlation based flow-field measurements. The approaches presented here are not limited to application in flow measurement, but could be useful for a variety of applications.

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Es wird ein "off-axis" Aufbau zur digitalholographischen Mikroskopie in Durchlicht- und Auflichtanordnung vorgestellt, der gleichzeitig hoch aufgelöste "full-field" Amplituden- und quantitative Phasenkontrastmikroskopie ermöglicht. Dabei werden verschiedene Algorithmen zur numerischen Rekonstruktion der komplexen Objektwelle bzgl. ihrer Eignung für die mikroskopische Anordnung miteinander verglichen. Durch Kombination eines beugungsfreien räumlichen Phasenschiebeverfahrens, das die Rekonstruktion ohne "Twin-Image" und nullte Beugungsordnung ermöglicht, und der Auswertung des Fresnel-Kirchhoff''schen Beugungsintegrals mit der Faltungsmethode werden die besten Ergebnisse erzielt. Die gleichzeitige Rekonstruktion der Amplitude und der Phase der Objektwelle aus einem einzigen Hologramm erfordert die mathematische Beschreibung der räumlichen Phasenverteilung in der Hologrammebene. Zur Bestimmung der Modellparameter wurde ein effizienter Algorithmus entwickelt und hinsichtlich seiner Genauigkeit getestet. Darüber hinaus wurde der Zusammenhang zwischen axialer Probenposition und dem Rekonstruktionsabstand, dessen Kenntnis zur quantitativen Auswertung und für eine rein numerische Fokussierung notwendig ist, hergeleitet. Anhand von Untersuchungen an technischen Objekten werden die laterale Auflösung und die Phasenauflösung des Systems quantifiziert und weitere experimentelle Parameter optimiert. Transparente biologische Proben, wie lebende Zellen, werden in Durchlichtanordnung analysiert. Dabei ist zur Bestimmung der Zelldicke die Kenntnis der Brechungsindizes von Zelle und Medium erforderlich. Hierfür wird ein experimentelles Verfahren vorgestellt, das die Abschätzung des integralen Brechungsindexes von Einzelzellen anhand ihrer rekonstruierten räumlichen Phasenverteilung ermöglicht. Exemplarisch wird Zelldifferenzierung aufgrund morphologischer Eigenschaften nachgewiesen und es werden Ergebnisse dynamischer Untersuchungen an lebenden Zellen gezeigt und diskutiert. / An off-axis setup for digital holographic microscopy in incident and transmission light arrangement for simultaneous high resolution full field amplitude and quantitative phase contrast microscopy is presented. Different kinds of algorithms for numerical reconstruction of the complex object wave are compared concerning their applicability to the microscopy arrangement. By combining a non-diffractive spatial phase shifting algorithm that performs reconstruction without the disturbing terms twin image and zero order with the numerical evaluation of the Fresnel-Kirchhoff diffraction integral by a convolution method we achieve best results. The simultaneous reconstruction of the object wave''s amplitude and phase from a single hologram requires a mathematical model of the spatial phase distribution within the hologram plane. An efficient numerical algorithm has been developed for determining the model''s parameters automatically and tested concerning its accuracy. Furthermore, the relation between the axial position of the object and the distance of reconstruction which is required for the quantitative evaluation of the reconstructed images and the application of a pure numerical focus is derived. Technical objects were used to quantify the lateral resolution and the phase resolution of the system and to optimize several parameters of the setup. Biological probes such as living cells are analyzed in transmission light arrangement. As a result the knowledge of the refractive index of the medium and the cell is required to derive the cell''s thickness from the reconstructed phase. Thus a special experimental method for the approximation of the integral refractive index of single cells from the reconstructed phase has been developed. Finally results of cell differentiation by morphological varieties as well as results of stimulated dynamic morphological changes are presented and discussed.