Digital holographic microscopy is an interferometric imaging technique, the principle of which is the off-axis image plane holography. The principle of this technique enables to reconstruct both the image intensity and the image phase from the output interferencesignal. The reconstruction can be carried out on the basis of a single image plane hologram. This leads to the possibility of a realtime image reconstruction. The speed of the reconstruction depends on the detection and the computing process. The aim of this diploma thesis is to develop user software for the control of the detection camera and for the image plane hologram reconstruction. The effort was to achieve the highest number of image reconstructions per time unit, with the maximum utilization of the data transfer between the camera and the computer.The next aim of this thesis is the stabilization of the optical table position. The method of stabilization is based on the image phase information, which is used for the control loop feedback between reconstructed image phase and the piezoelectric actuator placed inside of the optical table. Experimental results, which prove the functionality of the stabilization, are presented.

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 focuses on the image reconstruction from holographic microscope. The first part of the thesis summarizes problems of holography, describes its principle and application particularly in live cell biology. In the second part the description of several methods used for off-axis hologram reconstruction is provided. The last part describes the implementation of the basic step in holographic reconstruction – the elimination of autocorrelation and twin image – and then also the reconstruction of 3D information of the sample.

Tato dizertční práce se zabývá výrbou a charakterizací plasmonických nanostruktur. Její první část začíná krátkým úvodem do plasmoniky s navazujícím přehledem metod, které jsou v dnešní době nejčastěji používány k výrobě a charakterizaci plasmonických nanostruktur. Druhá část se pak zaměřuje na samotný výzkum, který byl v rámci PhD studia realizován. Cílem prvních experimentů bylo prozkouat možnosti použití elektronové litografie za variabilního tlaku v procesní komoře pro výrobu plasmonických nanostruktur na nevodivých substrátech jako je např. sklo. Jelikož se jedná o materiály, které jsou velice často používány k přípravě plasmonických struktur pacujících v oblasti viditelného světla. Druhá sekce pak diskutuje některé specifické aspekty přípravy plasmonických mikrostruktur elektronovou litografií pro THz oblast. Poslední část se pak zaměřuje na funkční vlastnosti plasmonických nanostruktur, převážně pak na kvantitativní charakterizaci fáze dalekého pole indukovaného plasmonickými nanostrukturami a jejich aplikacemi v oblasti optických metapovrchů - uměle připravených povrchů, které mohou být použity jako planární optické komponenty. Práce demonstruje a diskutuje různé experimentální přístupy použití mimoosové holografické mikroskopie pro jejich charakterizaci.

Digital holographic microscopy (DHM) is noninvasive method for obtaining images even from samples with low contrast. Nowadays DHM design makes it possible to illuminate sample by broad light source, halogene lamp. Broad light source is displayed in the front focal plane of condensor in such way, that Köhler illumination is achieved. Each point of the source corresponds to a plane wave in image field of objective, which illuminates the whole field of view, but from different direction. Position of the point determines the direction of illumination. In this reason, the microscope enables so reconstruct not only intensity, but also phase of object wave. New designed and constructed interchangeable pinhole aperture modify broad light illumination by rotation around the optical axis. Aperture is placed eccentrically considering the optical axis of microscope. Incidence of light beam on a sample would be under defined angle. After the reconstruction of taken phase images from individual angles of illumination should be possible to obtain three-dimensional structure of the sample.

Digital in-line holographic microscopy (DIHM) has been incorporated as an additional simultaneous in situ optical technique with ellipsomicroscopy for surface imaging and microscopy to study metastable pitting corrosion on stainless steel 316LVM in simulated biological solutions. DIHM adds microscopic volume imaging, allows one to detect local changes of the index of refraction in the vicinity of a pitting event, and allows one to track tracer particles and/or material ejected from the pitting sites. To improve the pitting corrosion resistance of stainless steel 316LVM, a simple surface treatment was tested and the aforementioned imaging techniques were used to verify that pitting occurred only on the wire face. Treatments consisted of polishing the samples to remove the passive layer, then immersing the wires in 90 C nanopure water for several hours. Treated wires show a marked increase in pitting corrosion resistance over untreated wires: the pit initiation potential increases by a minimum of 200 mV. Additional testing with scanning electron microscopy and energy dispersive X-ray spectroscopy indicate that the removal of sulphide inclusions from the surface is the most probable cause of this enhancement. To increase holographic reconstruction performance, Graphics Processing Units (GPUs) have been used; 4 Mpixel holograms are reconstructed using the dot product approximation of the Kirchhoff-Fresnel integral in 60 ms on a Tesla c1060 GPU. Errors in sizes and positions can easily be as large as 5 to 10 % for regions where the dot product approximation is not valid, so algorithms with fewer or no approximations are also required. Reconstructions for arbitrary holographic geometries using the full Kirchhoff-Fresnel integral take approximately 1 hour (compared to 1 week on a quad-core CPU), and reconstructions using convolution methods, in which the results of 256 reconstructions at 4096 x 4096 pixels in one plane are combined, take 17 s. This method is almost exact, with approximations only in the obliquity factor.

Moving towards label-free technologies is essential for many clinical and research applications. Raman spectroscopy is a powerful tool in the field of biomedicine for label-free cell characterisation and disease diagnosis, owing to its high chemical specificity. However, Raman scattering is a relatively weak process and can require long acquisition times, thus hampering its integration to clinical technologies. Multimodal analysis is currently pushing the boundaries in biomedicine, obtaining more information than would be possible using a single mode and overcoming any limitations specific to a single technique. Digital holographic microscopy (DHM) is a rapid and label-free quantitative phase imaging modality, providing complementary information to Raman spectroscopy, and is thus an ideal candidate for combination in a multimodal system. Firstly, this thesis explores the use of wavelength modulated Raman spectroscopy (WMRS), for the classification of immune cell subsets. Following this a multimodal approach, combining Raman spectroscopy and DHM, is demonstrated, where each technique is considered individually and in combination. The complementary modalities provide a wealth of information (both chemical and morphological) for cell characterisation, which is a step towards achieving a label-free technology for the identification of human immune cells. The suitability of WMRS to discriminate between closely related neuronal cell types is also explored. Furthermore optical spectroscopic techniques are useful for the analysis of food and beverages. The use of Raman and fluorescence spectroscopy to successfully discriminate between various whisky and extra-virgin olive oil brands is demonstrated, which may aid the detection of counterfeit or adulterated samples. The use of a compact Raman device is utilised, demonstrating the potential for in-field analysis. Finally, monodisperse and highly spherical nanoparticles are synthesised. A short study demonstrates the potential for these nanoparticles to benefit the techniques of surface enhanced Raman spectroscopy and optical trapping, by way of minimising variability.

The main objective of this thesis is the development of new processing techniques for digital holograms. The present work is part of the HoloFlow project that intends to integrate the DHM technology for the monitoring of water quality. Different tools for an automated analysis of digital holograms have been developed to detect, refocus and classify particles in continuous fluid flows. A detailed study of the refocusing criterion permits to determine its dependencies and to quantify its robustness. An automated detection procedure has been developed to determine automatically the 3D positions of organisms flowing in the experiment volume. Two detection techniques are proposed: a usual method based on a global threshold and a new robust and generic method based on propagation matrices, allowing to considerably increase the amount of detected organisms (up to 95 %) and the reliability of the detection. To handle the case of aggregates of particles commonly encountered when working with large concentrations, a new separation procedure, based on a complete analysis of the evolution of the focus planes, has been proposed. This method allows the separation aggregates up to an overlapping area of around 80 %. These processing tools have been used to classify organisms where the use of the full interferometric information of species enables high classifier performances to be reached (higher than 93 %). / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Práce se zabývá interpretací kvantitativního fázového zobrazení pomocí techniky koherencí řízené holografické mikroskopie. Vzhledem k tomu, že tato technika generuje velké množství kvantitativních fázových obrazů o nezanedbatelné velikosti, manuální analýza by byla časově náročná a neefektivní Za účelem urychlení analýzy obrazů získaných pomocí koherencí řízené holografické mikroskopie je v této práci navržena metodika automatizované interpretace kvantitativních fázových obrazů pomocí strojového učení s učitelem. Kvantitativní fázové obrazy umožňují extrakci parametrů charakterizujících distribuci suché hmoty v buňce a poskytují tak cennou informaci o buněčném chování. Cílem této práce je navrhnout metodologii pro automatizovanou klasifikaci buněk při využití této kvantitativní informace jak ze statických, tak z časosběrných kvantitativních fázových obrazů. Navržená metodika byla testována v experimentech s živými buňkami, jimiž byla vyhodnocena výkonnost klasifikace a významnost parametrů získaných z kvantitativních fázových obrazů.

This master’s thesis deals with the design of methodology for measurement of refractive index and thickness of living cells by coherence-controlled holographic microscope. The theoretical part summarises the holographic microscopy and its development at IPE FME BUT in Brno. The thesis focuses on the multimodal holographic microscope, its description, the principle, the procedure of work and data processing. Confocal microscopy is also described, which serves to compare the acquired values with the proposed methodology. The last part of the theoretical part deals with the testing of statistical hypotheses, which is needed for the processing of measured data. Experiments were designed for the verification of methodology for determination of the refractive index and cell thickness. The experimental part of the thesis deals with the sample preparation and measurement. The procedure and results of the proposed experiments and their evaluation follows.