Optical imaging is the imaging of objects with visible light. It is a tool often used for diagnostic purposes, such as in biomedical and material sciences. Digital holography is an optical imaging technique that captures and images the amplitude as well as the phase (the complex amplitude) of the lightwave. An advantage is that the complex amplitude can be calculated in different propagation planes. The goal of this thesis is to use digital holography to image depth of a reflecting surface aswell asmake 3D images of biological samples, and to contribute to the theoretical understanding in this regard. @en

We demonstrate large scale polarization contrast optical diffraction tomography (ODT). In cross-polarized sample arm detection configuration we determine, from the amplitude of the optical wavefield, a relative measure of the birefringence projection. In parallel-polarized sample arm detection configuration we image the conventional phase projection. For off-axis sample placement we observe for polarization contrast ODT, similar as for phase contrast ODT, a strongly reduced noise contribution. In the limit of small birefringence phase shift δ we demonstrate tomographic reconstruction of polarization contrast images into a full 3D image of an optically cleared zebrafish. The polarization contrast ODT reconstruction shows muscular zebrafish tissue, which cannot be visualized in conventional phase contrast ODT. Polarization contrast ODT images of the zebrafish show a much higher signal to noise ratio (SNR) than the corresponding phase contrast images, SNR=73 and SNR=15, respectively.

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In this work we demonstrate large-scale high-sensitivity optical diffraction tomography (ODT) of zebrafish. We make this possible by three improvements. First, we obtain a large field of view while still maintaining a high resolution by using a high magnification over numerical aperture ratio digital holography set-up. With the inclusion of phase shifting we operate close to the optimum magnification over numerical aperture ratio. Second, we decrease the noise in the reconstructed images by implementing off-axis sample placement and numerical focus tracking in combination with the acquisition of a large number of projections. Although both techniques lead to an increase in sensitivity independently, we show that combining them is necessary in order to make optimal use of the potential gain offered by each respective method and obtain a refractive index (RI) sensitivity of 8⋅10 ⁻⁵ . Third, we optimize the optical clearing procedure to prevent scattering and refraction to occur. We demonstrate our technique by imaging a zebrafish larva over 13 mm ³ field of view with 4 micrometer resolution. Finally, we demonstrate a clinical application of our technique by imaging an entire adult cryoinjured zebrafish heart.

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In this work we demonstrate large scale high sensitivity optical diffraction tomography (ODT) of zebrafish. Compared to previous work the scale and sensitivity are enhanced by the following steps. First, we obtain a large field of view while still maintaining a high image resolution by using a high magnification over numerical aperture ratio ODT set-up. With the inclusion of phase shifting we demonstrate that we operate close to the optimum magnification over numerical aperture ratio. Second, we decrease the noise in the reconstructed images by implementing off-axis sample placement and numerical focus tracking in combination with the acquisition of a large number of projections. Although both techniques lead to an increase in sensitivity independently, we show that combining them is necessary in order to make optimal use of the potential gain offered by each respective method and obtain a refractive index (RI) sensitivity of 8•10^-5. In this way, high RI sensitivity can be achieved that is necessary for phase tomography of optically cleared tissue structures, which we can identify for features with RI down to 6•10^-4. Third, we optimize the optical clearing procedure to prevent scattering and refraction to deteriorate our large scale images. We demonstrate our technique by imaging a 3 day old zebrafish and an adult cryoinjured zebrafish heart in a large 5.5 x 5.5 x 4.1 mm³ volume with 4 micrometer resolution. Various tissue structures can be clearly identified. The volume of the cryoinjured heart is segmented and quantified based on the refractive index distribution.

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Corneal endothelium images obtained by in vivo specular microscopy provide important information to assess the health status of the cornea. Estimation of clinical parameters, such as cell density, polymegethism, and pleomorphism, requires accurate cell segmentation. State-of-the-art techniques to automatically segment the endothelium are error-prone when applied to images with low contrast and/or large variation in cell size. Here, we propose an automatic method to segment the endothelium. Starting with an oversegmented image comprised of superpixels obtained from a stochastic watershed segmentation, the proposed method uses intensity and shape information of the superpixels to identify and merge those that constitute a cell, using Support Vector Machines. We evaluated the automatic segmentation on a dataset of in vivo specular microscopy images (Topcon SP-1P), obtaining 95.8merged cells and 2.0the parameter estimation against the results of the vendor’s builtin software, obtaining a statistically significant better precision in all parameters and a similar or better accuracy. The parameter estimation was also evaluated on three other datasets from different imaging modalities (confocal microscopy, phasecontrast microscopy, and fluorescence confocal microscopy) and tissue types (ex vivo corneal endothelium and retinal pigment epithelium). In comparison with the estimates of the datasets’ authors, we achieved statistically significant better accuracy and precision in all parameters except pleomorphism, where a similar accuracy and precision were obtained.

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Despite a substantial genetic component, efforts to identify common genetic variation underlying depression have largely been unsuccessful. In the current study we aimed to identify rare genetic variants that might have large effects on depression in the general population. Using high-coverage exome-sequencing, we studied the exonic variants in 1265 individuals from the Rotterdam study (RS), who were assessed for depressive symptoms. We identified a missense Asn396Ser mutation (rs77960347) in the endothelial lipase (LIPG) gene, occurring with an allele frequency of 1% in the general population, which was significantly associated with depressive symptoms (P-value=5.2 × 10 -08, β=7.2). Replication in three independent data sets (N=3612) confirmed the association of Asn396Ser (P-value=7.1 × 10 -03, β=2.55) with depressive symptoms. LIPG is predicted to have enzymatic function in steroid biosynthesis, cholesterol biosynthesis and thyroid hormone metabolic processes. The Asn396Ser variant is predicted to have a damaging effect on the function of LIPG. Within the discovery population, carriers also showed an increased burden of white matter lesions (P-value=3.3 × 1 -02) and a higher risk of Alzheimer's disease (odds ration=2.01; P-value=2.8 × 10 -02) compared with the non-carriers. Together, these findings implicate the Asn396Ser variant of LIPG in the pathogenesis of depressive symptoms in the general population.

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We present sub-millimeter full-field depth from focus digital holography of surface topography of rough objects. For each pixel, the depth of the object is calculated from the variance of the intensity image over a set of reconstruction distances. First, we theoretically describe the axial resolution of this method and show that sub-millimeter resolution is feasible. Second, using a digital holography setup without magnifying optics or lateral scanning we experimentally demonstrate 100 μm axial resolution depth ranging and surface topography imaging. This is significantly better than what has previously been reported using digital holography and could make this technique useful for rapid large-area characterization of surface topography of objects.@en

We use digital holography to quantify surface topography of rough objects in full-field. We calculate the variance of the intensity image as a focus metric over a set of reconstruction distances for each pixel, which results in a focus metric curve. The distance where the variance peaks is an estimate for the depth. First we analyze the lateral resolution of this method using the Talbot effect and argue that sub-mm axial resolution is feasible. Then, using a Michelson setup without magnifying optics or lateral scanning we experimentally demonstrate that sub-mm FWHM width of the focus curve can be achieved. This is significantly better than what was previously reported using digital holography and could make this technique useful for characterising objects in art and machine vision.@en