Cette thèse a pour objectif l’étude d’un lien effectif potentiel entre la motilité cellulaire, la mécanique cellulaire, et l’activité biochimique de ces mêmes cellules. Ce couplage a été étudié dans divers systèmes biologiques, et aussi bien dans des cultures de cellules qu’à l’intérieur de tissus plus complexes. Notamment, nous avons particulièrement cherché à détecter un couplage électromécanique dans des neurones qui pourrait être impliqué dans la propagation du message nerveux.Pour ce faire, nous avons dû développer deux microscopes optiques à la sensibilité extrême. Ces microscopes se composent de deux parties principales. La première sert à détecter des mouvements axiaux plus petits que la longueur d’onde optique, soit en dessous de 100 nanomètres. La deuxième partie permet la détection d’un signal de fluorescence, offrant la possibilité de suivre l’évolution biochimique de la cellule. Avec ces deux microscopes multimodaux, il est donc possible de suivre de manière simultanée un contraste de motilité, un contraste mécanique, un contraste structurel et un contraste biochimique. Si l’un de ces systèmes est basé sur la tomographie de cohérence optique plein champ et permet de faire de telles mesures en 3-D et en profondeur dans les tissus biologiques, le second ne permet que des mesures dans des cultures de cellules, mais est bien plus robuste au bruit mécanique. Dans ce manuscrit, nous allons essentiellement décrire le développement de ces deux appareils, et préciser les contrastes auxquels ils sont sensibles spécifiquement.Nous développerons également deux des applications principales de ces microscopes que nous avons étudié dans le détail au cours de cette thèse. La première application développe l’intérêt d’un de nos microscopes pour la détection sans marquage des principaux composants cellulaires et structuraux de la cornée et de la rétine. La seconde application tend à détecter et à suivre des ondes électromécaniques dans des neurones de mammifères / This PhD project aims to explore the relationship that might exist between the dynamic motility and mechanical behavior of different biological systems and their biochemical activity. In particular,we were interested in detecting the electromechanical coupling that may happen in active neurons, and may assist in the propagation of the action potential. With this goal in mind, we have developed two highly sensitive optical microscopes that combine one modality that detects sub-wavelength axial displacements using optical phase imaging and another modality that uses a fluorescence path. Therefore, these multimodal microscopes can combine a motility, a mechanical,a structural and a biochemical contrast at the same time. One of this system is based ona multimodal combination of full-field optical coherence tomography (FF-OCT) and allows the observation of such contrast inside thick and scattering biological tissues. The other setup provides a higher displacement sensitivity, but is limited to measurements in cell cultures. In this manuscript, we mainly discuss the development of both systems and describe the various contrastst hey can reveal. Finally, we have largely used our systems to investigate diverse functions of the eye and to look for electromechanical waves in cell cultures. The thorough description of both biological applications is also provided in the manuscript

Tato diplomová práce se zabývá výzkumem na téma vlivu prostorové koherence osvětlení. Účelem je určit schopnost osové lokalizace při zobrazení Koherencí řízeným holografickým mikroskopem (CCHM) v závislosti na různé prostorové koherenci světelného zdroje. Osová lokalizace je v tomto případě zkoumána jako kvalita rozlišení drobných detailů trojrozměrného vzorku, umístěných nad sebou. Teorie zobrazení holografickým mikroskopem a teorie rozptylu v nehomogenních prostředích je shrnuta v první části práce, v rozsahu nutném pro pochopení části praktické. Základní princip fungování mikroskopu a přesný popis jeho uspořádání je zde podrobně popsán. Proběhl mechanický návrh stavební úpravy mikroskopu tak, aby bylo možno využívat kondenzorovou optiku s vysokou numerickou aperturou a omezenými optickými vadami. Několik různých přístupů, které by mohly vést ke zlepšení zobrazovacích vlastností mikroskopu, bylo navrženo a vyzkoušeno a jsou zde popsány i s jejich výhodami a nevýhodami. Pro experimentální část práce byl vyroben modelový vzorek. Závislost osové lokalizace na prostorové koherenci osvětlení byla demonstrována pomocí simulace a následně ověřena experimentálně, pozorováním vyrobeného modelového vzorku. Experimentální výsledky potvrzují základní principy vycházející ze zmíněné teorie. Na závěr jsou navržena možná vylepšení, pro budoucí zpřesnění výsledků.

This diploma thesis deals with the issue of motility analysis in leukemia cells. An accurate description of the cell movement and the detection of differences in motility under experimental conditions can be obtained by quantitative analysis of cell motility using time-lapse recording. The first part of this work describes various types of tumor cell migraton. The second part focuses on methods of analysis of cell motility in tissue culture using time-lapse recording, which include image acquisition and processing. Part of this chapter describes a coherence-controlled holographic microscope, which was used in the practical part and for which an insert was designed to ensure the exact and stable position of the individual chambers. The last part is focused on the research of leukemic cell motility, which is concluded by a discussion of the obtained results. The appendix contains a published study included acknowledgement to the author of this diploma thesis for participation in the project.

The diploma thesis deals with the registration of images taken with the multimodal holographic microscope (MHM). The summary covers the fluorescent and holographic microscopy, and the multimodal holographic microscope combining both these microscopy types. Every pair of the images needs to be aligned in order to gain new information by combining both image types. The thesis contains an algorithm that registers images by phase correlation as well as a process created in MATLAB in accordance with the algorithm. The most important procedure parameters’ influence on the registration success is described and the results are annotated.

<div>Vibrational spectroscopic imaging has become an emerging platform for chemical visualization of biomolecules and materials in complex systems. For over a century, both Raman and infrared spectroscopy have demonstrated the capability to recognize molecules of interest by harnessing the characteristic features from molecular fingerprints. With the recent development of hyperspectral vibrational spectroscopy imaging, which records the chemical information without sacrificing the spatial-temporal resolution, numerous discoveries has been achieved in the field of molecular and cellular biology. Despite the ability to provide complimentary chemical information to Raman-based approaches, infrared spectroscopy has not been extensively applied in routine studies due to several fundamental limitations: 1). the poor spatial resolution; 2). inevitable strong water absorption; 3). lack of depth resolution.</div><div>Mid-infrared photothermal (MIP) microscopy overcame all the above mentioned problems and for the first time, enabled depth-resolved in vivo infrared imaging of live cells, microorganisms with submicrometer spatial resolution. The development of epi-detected MIP microscopy further extends its application in pharmaceutical and materials sciences. With the deployment of difference frequency generation and other nonlinear optical techniques, the spectral coverage of the MIP microscopy was significantly enhanced to enable chemical differentiation in complex systems across the broad mid-infrared region. In addition to the efforts to directly improve the performance of MIP microscopy, a novel quantitative phase imaging approach based on polarization wavefront shaping via custom-designed micro-retarder arrays was developed to take advantage of the highly sensitive phase measurement in combination with the photothermal effect. Besides, the extended depth-of-field and multifocus imaging enabled by polarization wavefront shaping could both improve the performance of MIP microscopy for volumetric imaging.</div>

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This thesis presents a new optical imaging technique which measures the structure of objects without the use of lenses. Termed diffraction-contrast imaging (DCI), the method retrieves the object structure from a Fresnel diffraction pattern of the object, using a deconvolution algorithm. DCI is particularly adept at imaging highly transparent objects and this is demonstrated by retrieving the structure of an almost transparent cloud of laser-cooled atoms. Applied to transparent Bose-Einstein condensates, DCI should allow the non-destructive imaging of the condensate while requiring only the minimum possible apparatus of a light source and a detector. (For complete abstract open document)

This doctoral thesis deals with biophysical interpretation of quantitative phase imaging (QPI) gained with coherence-controlled holographic microscope (CCHM). In the first part methods evaluating information from QPI such as analysis of shape and dynamical characteristics of segmented objects as well as evaluation of the phase information itself are described. In addition, a method of dynamic phase differences (DPD) is designed to allow more detailed monitoring of cell mass translocations. All of these methods are used in biological applications. In an extensive study of various types of cell death, QPI information is compared with flow cytometry data, and preferably a combination of QPI and fluorescence microscopy is used. The DPD method is used to study mass translocations inside the cell during osmotic events. The simplified DPD method is applied to investigate the mechanism of tumor cell movement in collagen gels.

The dissertation deals with the image processing in digital holographic microscopy and X-ray computed tomography. The focus of the work lies in the proposal of data processing techniques to meet the needs of the biological experiments. Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The phase images are affected by the phase aberrations that make the analysis particularly difficult. Here, we present a novel algorithm for dynamical processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. High resolution X-ray computed tomography is increasingly used technique for the study of the small rodent bones micro-structure. In this part of the work, the trabecular and cortical bone morphology is assessed in the distal half of rat femur. We developed new method for mapping the cortical position and dimensions from a central longitudinal axis with one degree angular resolution. This method was used to examine differences between experimental groups. The bone position in tomographic slices is aligned before the mapping using the propound standardization procedure. The activity of remodelling process of the long bone is studied on the system of cortical canals.