The new arrangement of the off-axis holographic module, which is using polarizationactive diffraction grating divides signal into reference and subject wave of an interferometer based on their polarization. However, current design of the module does not have a possibility to tune a length of the optical paths. Thus the inability to tune optical paths leads to a reduced quality of interference structure during observation of biological samples. The current module is only suitable for technical applicating due to this limitation. Possibility of tuning branches is key step in biological applications. Therefore a new computer-controlled module is created in order to enable use for biological samples.

L’étude de phénomènes thermiques à l’échelle microscopique peut s’avérer compliquée à mettre en place, principalement à cause de l’absence de technique de mesure de température fiable. Dans ce contexte, une technique de mesure de température appelée TIQSI a été développée au sein de l’Institut Fresnel. Dans l’objectif d’étudier des phénomènes thermo-induit à l’échelle microscopique, j’ai monté un microscope capable de contrôler et de quantifier une élévation de température à l'aide de TIQSI et de nanoparticules d’or. Différents phénomènes ont ainsi pu être étudiés.La synthèse hydrothermale regroupe les réactions chimiques utilisant de l’eau liquide à des températures plus élevées que la température d’ébullition. L’utilisation de nanoparticules permet d’avoir de l’eau liquide à des températures supérieures à 100°C (état métastable). J’ai pu ainsi effectuer des réactions de synthèse hydrothermale sans autoclave ce qui constitue un nouveau concept en chimie de synthèse.Une cellule vivante peut-être endommagée par un stress de chaleur ce qui peut détériorer ses protéines. En réponse à ce stress, la synthèse de HSP permet la réparation des protéines endommagées. J’ai pu étudier la dynamique de réponse des HSP ce qui a permis d’illustrer l’intérêt d’une chauffe locale et de TIQSI pour ce genre d’expérience.Une autre application mêlant le surchauffage de l’eau liquide et la biologie a été abordée. Les organismes hyperthermophiles vivent à de très hautes températures (80-110◦C). J’ai pu durant mes expériences observer le déplacement d’hyperthermophiles. Cette avancée constitue les prémices d’expériences plus ambitieuses comme l’étude de l’interaction entre hyperthermophiles. / Nowadays, thermal experiments at the microscopic scale remain challenging to conduct due to the lack of reliable temperature measurment techniques. To solve these problems, a label-free temperature measurement technique called TIQSI has been developed in the Institut Fresnel.With the objective to study new thermal-induced effects on the microscale using TIQSI, I built a microscope aimed to control heat diffusion on the microscale using nanoparticle. Thus, I could study different phenomena in chemistry and biology.Hydrothermal methods in chemical synthesis rely on the use of superheated liquid water as a solvent. It has been shown that gold nanoparticles can be used superheated water in a metastable state. I managed to conduct hydrothermal chemistry experiments using thermoplasmonics without autoclave which represents a new paradigm in chemistry.A living cell can be damaged by a heat stress which can misfold its proteins. To response to this stress, the HSP synthesis enables the reparation of misfolded proteins. I could study the heat stress response of HSP at short time scale which allowed me to illustrate the interest of using TIQSI and a local heat.As an application mixing superheating water and biology, I studied organisms that are able to live at high temperature (80-110°C) namely hyperthermophiles. Motion of these organisms has been studied without autoclave which paves the way to more sophisticated experiments such as the interaction between hyperthermophiles.

This doctoral thesis deals with design of a new generation of coherence-controlled holographic microscope (CCHM). The microscope is based on off-axis holographic configuration using diffraction grating and allows the use of temporally and spatially incoherent illumination. In the theoretical section a new optical configuration of the microscope is proposed and conditions for different parameters of the microscope and its optical components are derived. The influence of different sources of noise on phase detection sensitivity is studied. In the next section design of experimental setup is described and automatable adjustment procedure is proposed. Last section describes experimental verification of the most important optical parameters of the experimental setup. When compared to previous generation of CCHM, the newly proposed configuration uses infinity-corrected objectives and common microscope condensers, allows more space for the specimens, eliminates the limitation of spectral transmittance and significantly simplifies the adjustment procedure so that automation of this procedure is possible.

Concrete, one of the world’s most important building materials is formed when cement is mixed with aggregates and reacts with water. The reaction is called hydration. Production of cement involves conversion of limestone and clay minerals into cement clinker in a kiln at high temperatures. The process requires high amounts of energy and causes substantial carbon emissions due to calcination of limestone and combustion of fuels, and the need for carbon neutral clinker products have never been greater. Heidelberg Materials Cement Sverige AB has multiple ongoing projects to reduce the carbon footprint of their clinker products. In essence, this is made possible by diluting the clinker with supplementary cementitious materials (SCM), electrification of the kiln, and carbon capture and storage (CCS). During tests of such applications in pilot and industrial scales it is necessary to evaluate the cement clinker manufactured for its ability to act as a binder in concrete. Such properties are collectively termed hydraulic reactivity and depend on the rate and extent to which the anhydrous components (alite, belite, aluminate, ferrite) in the cement react with water to form structural strength. The primary hydration products are Calcium-silicate-hydrate (C-S-H) and portlandite (CH). Due to lack of routines for clinker reactivity testing, especially for small batches, new methods and guidelines for reactivity tests has become highly sought after. Therefore, this work has aimed to develop a method for laboratory grinding of clinkers and then to study their hydration reactions by isothermal conductive calorimetry (ICC), Rietveld refinement quantitative x-ray diffraction (XRD) and thermogravimetric analysis (TGA). The goal of the work has been to provide a grinding method for laboratory cement preparation and a protocol for clinker reactivity evaluation. A fundamental requirement has been that the results of the methods must be comparable with Heidelberg Materials’ conventional standard methods. The work was initiated with a literature review on cement clinker manufacture, its hydration kinetics and reactivity tests. Industrial reference clinkers were used to develop the grinding method, and finally, reactivity tests with ICC, XRD and TGA were conducted on multiple clinkers of different origins. The grinding method developed in this work gave a fineness resembling the conventional grinding method but slightly coarser. Consequently, the heats measured in ICC for the clinkers studied were also comparable to previous analyses by conventional methods. This was a direct result of the reactivity being dependent on the specific surface area of the cement particles. Thanks to this, the newly developed grinding method and reactivity test by ICC may be incorporated into Heidelberg Materials standard methods. Hydrates formation and clinker mineral consumption were studied in XRD and TGA. The two techniques were used as cross-validation of one another. In summary, these yielded more in-depth information about the hydration of cements than provided by ICC and gave insights into what minerals and reactions were responsible for each clinker’s reactivity. However, the XRD and TGA results contained significant errors at some times, and further development is necessary before using them as part of a standard routine. This was mainly due to errors tied to sample preparation. Some necessary improvements are better Rietveld refinement, prevention of XRD sample carbonation, and addition of a separate ettringite analysis in TGA. Despite this, the methods show great promise, as highly correlating results were reached between methods when the sources of error were managed. For future work, it is suggested that the protocol is expanded and applied to also evaluate SCM’s. / Betong är ett av världens viktigaste byggmaterial och bildas när cement blandat med aggregat reagerar med vatten. Reaktionen kallas hydratation. Cement i sin tur tillverkas genom omvandling av kalksten och lermineraler till cementklinker vid hög temperatur i en roterugn. Denna process är mycket energikrävande och genererar stora koldioxidutsläpp från råmaterialen och bränslet. Till följd av detta har ett starkt och omedelbart behov av klimatvänliga klinkerprodukter uppstått. Heidelberg Materials Cement Sverige AB arbetar ständigt med att hitta nya lösningar till produktionen för att minska dess klimatavtryck. En viktig del i denna minskning är att späda ut klinkern med alternativa bindemedel (SCM), elektrifiering av bränningsprocessen och uppfångning samt lagring av koldioxid (CCS). Förändringar i den industriella processen förändrar dock klinkern vilket kan påverka cementets förmåga att agera som bindemedel i betong. Dessa egenskaper kallas kollektivt för hydraulisk reaktivitet, och beror på både hastigheten och i vilken utsträckning klinkermineralerna alit, belit, aluminat och ferrit hydratatiseras för att bilda calcium-silikat-hydrat (C-S-H) och portlandit (CH) och på så vis skapa tryckhållfasthet. I och med försök på industriell- och pilotnivå har det blivit önskvärt att kunna utvärdera reaktiviteten hos klinker. Eftersom det idag delvis fattas rutiner för detta, så har detta arbete syftat till att utveckla en metod för laboratoriemalning av klinker i små batcher, samt att studera hydratationen av den malda klinkern med isotermisk konduktions kalorimetri (ICC), kvantitativ röntgendiffraktion med Rietveld metoden (XRD) och termogravimetrisk analys (TGA). Projektets mål har varit att färdigställa en sådan malningsmetod och att förse företaget med ett protokoll för utvärdering av reaktiviteten. Ett grundläggande krav för de utvecklade metoderna är att deras resultat ska vara jämförbara med Heidelberg Materials konventionella standardmetoder. Arbetet sjösattes med en litteraturstudie på ämnet klinkerproduktion, cementhydratation och reaktivitetstester av cement och alternativa bindemedel. Därefter utvecklades malningsmetoden med hjälp av industriell referensklinker. Slutligen testades dessa och ett antal andra klinkers, både framställda i laboratorieugn och industriellt, för reaktivitet med ICC, XRD och TGA. Malningsmetodens resultat blev något grövre, men ändå i hög grad jämförbart med dagens konventionella metod. Denna skillnad är dock liten och förutsägbar. Därför blev också uppmätt värme i ICC jämförbart med tidigare värden från den konventionella metoden. På grund av den något grövre malningen, vilket leder till mindre reaktionsyta för cementet-vatten-fasen, så blev värmeutvecklingen i ICC alltid något lägre jämfört med den konventionella metoden, dock aldrig utanför gränserna för vad standardmetodens reproducerbarhet är. Tack vare detta dras slutsatsen att protokollet kommer vara relevant och lämpligt för introduktion i industrin. Cementens reaktivitet studerades också i högre detalj med hjälp av TGA och XRD, vilka användes för extern validering av varandra. Medan dessa metoder ger viktig information om varje enskild fas i den åldrande pastan, så är slutsatsen att de är i fortsatt behov av utveckling. Detta har mest att göra med provberedningen. Nödvändiga förbättringar är bättre Rietveld kvantifiering med fler prover och försiktigare provberedning för att förhindra karbonatisering av cementpastorna. TGA metoden kan enkelt förbättras och uppnå avsevärt bättre resultat endast genom införandet av en separat analys av ettringit. Trots detta så visar de två metoderna hög korrelation mellan varandra då provberedningen fungerat som avsett, vilket är lovande och innebär att man med dessa enkla förslag kan skapa en metod som ger information om ett cements reaktivitet i mycket högre detalj än vad som är möjligt med dagens standardmetoder. Som förslag till framtida arbeten ges att protokollet bör utökas till att även bedöma prestandan av alternativa bindemedel vid spädning av klinker. / Cemzero

[pt] A análise de sistemas de medição consiste do exame da adequação do sistema quanto ao operador, ao instrumento e a outras fontes de variação, bem como da comparação da variância do erro de medição com a variância natural do processo. Nesse aspecto, esta pesquisa teve por objetivo a análise das incertezas da quantificação de fase pelo método de Rietveld em análise de pó. Os efeitos de vários fatores na quantificação de fase foram avaliados utilizando técnicas estatísticas de planejamento experimental e de análise multivariada, com a utilização de materiais de alto nível de rastreabilidade na realização dos experimentos, no Laboratório de difração de raios X do Departamento de Ciências de Materiais e Metalurgia da PUC-Rio. Através da determinação do construto de variação do processo, constatou-se que a quantificação das fases analisadas sofre influência das condições de medição de forma diferenciada de material para material, impossibilitando a obtenção de uma fórmula geral para cálculo dos erros de quantificação, embora os erros possam ser determinados por uma análise de repetitividade e reprodutibilidade apropriadamente conduzida. / [en] The analysis of measurement systems is done by the examination of the adequacy of the system according to the operator, the instrument and other sources of variability, as well as by the comparison of the measurement error variance with the natural process variance. This research consisted in the evaluation of the uncertainties of phase quantification in powder analysis by the Rietveld method. The evaluation of the effects of several factors on the phase quantification was performed using statistical techniques of design of experiments and of multivariate analysis, with the use of materials of high level of traceability for the conduction of the experiments, in the Laboratory of X-ray Diffraction of the Pontifícia Universidade Católica do Rio de Janeiro. Through the determination of the variational structure of the process it was verified that the analyzed phases suffer influence of the measurement conditions in a differentiated way, which prevents the obtention of a general formula for calculation of the quantification error, although the errors can be determined by a repeatability and reproducibility analysis properly conducted.

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>