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.

This master’s thesis develops a novel method of digital holography, from recent studies known as Fresnel Incoherent Correlation Holography (FINCH). The method enables the reconstruction of the correlation records of three-dimensional objects, captured under quasi-monochromatic, incoherent illumination. The experimental system is based on an action of a Spatial Light Modulator, driven by computer generated holograms to create mutually correlated beams. Both optical and digital parts of the experiment can be carried out using procedures of classical holography, diffractive optics and digital holography. As an important theoretical result of the master’s thesis, a new computational model was proposed, which allows to describe the experiment completely with respect to its two basic phases. The proposed model allows to understood the method intuitively and can be used additionally for analysis and interpretation of the imaging parameters and the system optimalization. The theoretical part of the master’s thesis also presents a detailed description of the correlation imaging based on an appropriate reconstruction process. Computational models were developed for both monochromatic and quasi-monochromatic illumination. In experimental part, all theoretical results were verified. The imaging parameters were examined using standard resolution target tests and appropriate biological samples. As an original experimental result, spiral modification of the system resulting in a vortex imaging was proposed and realized. Here, a selective edge enhancement of three-dimensional objects is possible, resulting in a significant extension of possible applications of the method.

Microscopic techniques with high spatial and temporal resolution are required for in vivo studying biological cells and tissues. Adaptive lenses exhibit strong potential for fast motion-free axial scanning. However, they also lead to a degradation of the achievable resolution because of aberrations. This hurdle can be overcome by digital optical technologies. We present a novel High-and-Low-frequency (HiLo) 3D-microscope using structured illumination and an adaptive lens. Uniform illumination is used to obtain optical sectioning for the high-frequency (Hi) components of the image, and nonuniform illumination is needed to obtain optical sectioning for the low-frequency (Lo) components of the image. Nonuniform illumination is provided by a multimode fiber. It ensures robustness against optical aberrations of the adaptive lens. The depth-of-field of our microscope can be adjusted a-posteriori by computational optics. It enables to create flexible scans, which compensate for irregular axial measurement positions. The adaptive HiLo 3D-microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 microns and sub-micron lateral resolution over the full scanning range. In result, volumetric measurements with high temporal and spatial resolution are provided. Demonstration measurements of zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are presented.

La microscopie par cohérence optique (OCM) est une technique d'imagerie tomographique basée sur l'interférométrie en lumière blanche permettant d'imager les milieux biologiques à l'échelle microscopique. L'OCM est une méthode particulièrement adaptée à l'imagerie dermatologique, en particulier pour le diagnostic du cancer de la peau, car elle permet d'obtenir des images similaires aux images histologiques sans nécessiter d'effectuer de biopsie.Ces travaux de thèse portent sur le développement de la microscopie par cohérence optique pour l'imagerie de la peau, dans le but de fournir au dermatologue un outil d'imagerie compact, adapté à l'imagerie dermatologique in vivo, et permettant d'obtenir des images à la fois structurelles et fonctionnelles.Un dispositif de microscopie par cohérence optique plein champ (FF-OCM) compact, à éclairage par LED blanche, a tout d'abord été développé, permettant d'obtenir des images tomographiques à très haute résolution (0.7 μm × 1.8 μm) jusqu’à ∼200 μm de profondeur dans la peau. En utilisant une LED de haute puissance, des images de peau in vivo ont pu être obtenues.A partir de ce dispositif de FF-OCM, des méthodes d'imagerie fonctionnelle permettant de cartographier les écoulements sanguins (angiographie) ont été mises en oeuvre. Quatre méthodes, basées sur une analyse du signal interférométrique (temporelle ou fréquentielle), d'images de phase ou d'images d'amplitude ont permis d'imager de l'intralipide s'écoulant dans un modèle de capillaire sanguin.L'imagerie fonctionnelle polarimétrique a aussi été explorée en FF-OCM. Une optimisation du contraste des images polarimétriques a été obtenue en modifiant les composants polarisants d'un montage conventionnel de FF-OCM polarimétrique en fonction de l'échantillon imagé. Cette méthode a été testée sur un échantillon polarisant simple.Finalement, une nouvelle méthode d'OCM, la microscopie par cohérence optique confocale à éclairage « ligne » (LC-OCM) a été étudiée, dans le but de développer un système permettant d'imager la peau in vivo, avec une plus grande profondeur de pénétration dans les tissus que la FF-OCM. Ce système, combinant un filtrage interférométrique et un filtrage confocal, a permis d'obtenir des images de peau in vivo en coupe verticale et en coupe en face, avec une résolution spatiale similaire à celle de la FF-OCM, mais à une profondeur supérieure atteignant 300 μm. / Optical coherence microscopy (OCM) is a technique for tomographic imaging based on white light interferometry, making it possible to image biological media with micrometer-scale spatial resolution. OCM is particularly well-suited to dermatological imaging, especially skin cancer diagnosis, since it provides images that are similar to histological images without the need for biopsy.This PhD thesis focuses on the development of OCM for skin imaging, with the aim of providing a compact, in vivo imaging tool for the dermatologist, capable of acquiring structural and functional images of the skin.A compact, full-field OCM (FF-OCM) system illuminated by a white LED was first developed, making it possible to obtain tomographic images at an ultra-high resolution (0.7 μm × 1.8 μm), up to ∼200 μm in depth within the skin. Using a high power LED, in vivo skin images could be obtained.Using this FF-OCM setup, functional imaging methods for blood flow mapping (angiography) were implemented. Four methods, based on temporal or frequency analysis of the interferometric signal, phase images or amplitude images, have been shown to be able to image intralipid flow within a model blood capillary.Functional polarimetric imaging has also been explored in FF-OCM. Contrast optimization in polarimetric images has been obtained by modifying the polarizing components of the conventional polarization sensitive FF-OCM setup depending on the sample to be imaged. This method has been tested on a simple polarizing sample.Finally, a new OCM method, line-field confocal OCM (LC-OCM), has been studied. The goal here was to develop a system capable of imaging the skin in vivo, with a tissue penetration depth greater than what is possible for FF-OCM. This system, which combines interferometric filtering and confocal filtering, makes it possible to obtain in vivo skin images in vertical and en face slices, with a spatial resolution similar to that of FF-OCM, but with a greater penetration depth of 300 μm.

Electrically tunable lenses exhibit strong potential for fast motion-free axial scanning in a variety of microscopes. However, they also lead to a degradation of the achievable resolution because of aberrations and misalignment between illumination and detection optics that are induced by the scan itself. Additionally, the typically nonlinear relation between actuation voltage and axial displacement leads to over- or under-sampled frame acquisition in most microscopic techniques because of their static depth-of-field. To overcome these limitations, we present an Adaptive-Lens-High-and-Low-frequency (AL-HiLo) microscope that enables volumetric measurements employing an electrically tunable lens. By using speckle-patterned illumination, we ensure stability against aberrations of the electrically tunable lens. Its depth-of-field can be adjusted a-posteriori and hence enables to create flexible scans, which compensates for irregular axial measurement positions. The adaptive HiLo microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 μm and sub-micron lateral resolution over the full scanning range. Proof of concept measurements at home-built specimens as well as zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are shown.

Electrically tunable lenses exhibit strong potential for fast motion-free axial scanning in a variety of microscopes. However, they also lead to a degradation of the achievable resolution because of aberrations and misalignment between illumination and detection optics that are induced by the scan itself. Additionally, the typically nonlinear relation between actuation voltage and axial displacement leads to over- or under-sampled frame acquisition in most microscopic techniques because of their static depth-of-field. To overcome these limitations, we present an Adaptive-Lens-High-and-Low-frequency (AL-HiLo) microscope that enables volumetric measurements employing an electrically tunable lens. By using speckle-patterned illumination, we ensure stability against aberrations of the electrically tunable lens. Its depth-of-field can be adjusted a-posteriori and hence enables to create flexible scans, which compensates for irregular axial measurement positions. The adaptive HiLo microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 μm and sub-micron lateral resolution over the full scanning range. Proof of concept measurements at home-built specimens as well as zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are shown.