To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.

Raman microscopy (RM) is a vibrational spectroscopic technique capable of obtaining sensitive measurements of molecular composition, structure, and dynamics from a very small sample volume (~1 µm). In this work, a RM system was developed for future applications in cellular radiobiology, the study of the effects of ionizing radiation on cells and tissues, with particular emphasis on the capability to investigate the internal molecular composition of single cells (10-50 µm in diameter). The performance of the RM system was evaluated by imaging 5 µm diameter polystyrene microbeads dispersed on a silicon substrate. This analysis has shown that RM of single cells is optimized for this system when using a 100x microscope objective and a 50 µm confocal collection aperture. Quantitative measurements of the spatial, confocal, and spectral resolution of the RM system have been obtained using metal nanostructures deposited on a flat silicon substrate. Furthermore, a spectral investigation of several substrate materials was successful in identifying low-fluorescence quartz as a suitable substrate for RM analysis of single cells. Protocols have been developed for culturing and preparing two human tumor cell lines, A549 (lung) and DU145 (prostate), for RM analysis, and a spectroscopic study of these two cell lines was performed. Spectra obtained from within cell nuclei yielded detectable Raman signatures from all four types of biomolecules found in a human cell: proteins, lipids, carbohydrates, and nucleic acids. Furthermore, Raman profiles and 2D maps of protein and DNA distributions within single cells have been obtained with micron-scale spatial resolution. It was also found that the intensity of Raman scattering is highly dependent on the concentration of dense nuclear material at the point of Raman collection. RM shows promise for studying the interactions of ionizing radiation with single cells, and this work has been successful in providing a foundation for the development of future radiobiological RM experiments.

Near-membrane and trans-membrane proteins and their interactions with the extracellular matrix (ECM) can yield valuable information about cell dynamics. However, advances in the field of nanoscale cellular processes have been hindered, in part, due to limits imposed by current technology. In this work, a novel evanescent field (EF) imaging technique is designed, modeled, created and tested for near-field imaging in the apical surface of cells. This technique and Förster resonance energy transfer (FRET) were used to investigate interactions between integrins on the cell surface and the ECM protein, fibronectin. The goal was to monitor changes in the integrin density at the cell surface as a function of clustering after binding to fibronectin on the microsphere surface. For the EF technique, quantum dot (QD)-embedded polystyrene microspheres were used to couple light into whispering gallery modes (WGMs) inside the microspheres; the resulting EF at the surface of the microsphere was used as a near-field excitation source with ~50 nm axial resolution for exciting fluorescently-labeled integrins. For FRET measurements (~10 nm axial resolution), QDs (donors) were coated on the surface of microspheres and energy transfer to red fluorescent protein (RFP)-integrin constructs (acceptors) studied. In both techniques, the QD-modified microspheres were mounted on atomic force microscope (AFM) cantilevers, functionalized with fibronectin, and brought into contact with fluorescently-labeled HeLa or vascular smooth muscle (VSM) cells. The results obtained from both methods show the clustering and activity of the integrins and are in good agreement with each other. Amsterdam discrete dipole approximation (ADDA) was used to study the effects of inhomogeneous surrounding refractive index on the quality factor and position of the WGMs due to the attachment of a microsphere to an AFM cantilever. WGMs of various QD-embedded microspheres mounted on AFM cantilevers were experimentally measured and shown to be consistent with the model.

A robotic microscope was designed using a microcontroller to take time-lapse digital photographs of developing salamander embryos. The microcontroller operated three stepper motors to control three-axis movement accurately, and two six mega-pixel digital cameras to capture through-focus time-lapse digital pictures of six views of Ambystoma mexicanum embryos (axolotl, a salamander). The device is designed to take images every five minutes for 80 hours of early development, from fertilization to stage 20, when the neural tube closes to form the brain and spinal column. Techniques to enhance the embryo images were investigated including image fusion to get in-focus views from a stack of images. In the early embryo surface epithelial cells differentiate to form neural tissue and external skin tissue. Observing the whole embryo surface at cellular level will give a better idea of the stress and strain each cell undergoes and what physical forces are involved in cell differentiation.

Die selbständige, gerichtete Bewegung von biologischen Zellen ist eine der grundlegendsten und komplexesten Erscheinungen der Natur. In höher entwickelten Lebewesen spielt die Zellbewegung eine wichtige Rolle, z.B. bei der Entwicklung des Organismus, bei der Funktion des Immunsystems aber auch bei der Metastase von Krebszellen. Die physikalischen Prozesse die dieser Fähigkeit zugrunde liegen, sind im Fokus dieser Arbeit. Um besser zu verstehen welche Prozesse im Einzelnen und in welcher Kombination den Zellen erlauben sich gerichtet fortzubewegen, wurde in der vorliegenden Arbeit ein representatives Modellsystem von motilen Zellen untersucht. Fischkeratozyten bewegen sich in vitro regelmäßig und gleichförmig, relativ schnell über die Substratfläche, und stellen aus physikalischer Sicht eine optimierte, sich selbständig bewegende Polymermaschine dar. Um Kräfte in der Bewegungsebene der Zellen zu untersuchen, wurde in der vorliegenden Arbeit eine neuartige, auf dem Rasterkraftmikroskop (RKM) basierende Methode entwickelt. Zusätzlich wurden hochaufgelöste, mit dem Phasenkontrastmikroskop aufgenommene Bilderserien analysiert und die Geschwindigkeitsverteilung in der Zelle durch Korrelationsalgorithmen bestimmt. Die Struktur des Polymernetzwerkes wurde in mit Fluoreszenzfarbstoff markierten Zellen untersucht, und elastische Eigenschaften wurden mit rheologischen RKM-Messungen bestimmt. Traktionskraftmessungen an elastischen Substraten runden das umfassende Bild ab. Durch Veränderung der molekularen Strukturen mit verschiedenen Chemikalien, die unterschiedliche Prozesse im Gesamtsystem stören, konnte nun ein Phasenraum der Kraftgenerierungsprozesse untersucht und unterschiedliche Effekte verschiedenen Prozessen eindeutig zugeordnet werden. Es wurde somit erstmalig experimentell bewiesen, dass die Polymerisation von Aktin die treibende Kraft am vorderen Rand der Zelle ist. Darüber hinaus wurde das Verhalten des Kraftaufbaus mit einem Model beschrieben, das Aufschluss über die Funktionsweise der darunterliegenden Aktinpolymerstrukturens gibt. Desweiteren wurde in der Mitte der Zelle, zwischen vorderem Rand und Zellkörper, erstmalig eine rückwärtsgerichtete Kraft gemessen, die wichtig ist um ein Kräftegleichgewicht zu erstellen. Ein Model das auf entropischen Kräften im Polymersystem basiert, beschreibt diese kontraktilen Kräfte und ordnet sie der Depolymerisation von Aktin zu. Die Bewegung des Zellkörpers wiederum basiert auf dem Zusammenspiel dieser beiden Mechanismen, sowie der Kontraktion von Aktin und Aktinbündeln durch molekulare Motoren. Eine umfassendes Charakterisierung über verschiedene lokale Mechanismen und ihrer Wechselwirkungen konnte somit erstellt werden, und damit das Verständnis der Kraftgenerierung zur Zellbewegung vertieft.

This work presents the novel applications of heated AFM tip in nanolithography. Different strategies were investigated for patterning materials using heated AFM tip. New materials were developed for these new nanolithography methods. Simulation and modeling work was done to further understand the heat transfer and chemical reactions involved in the thermal writing process. The selective thermal decomposition of polymer was the first thermal patterning method we¡¯ve investigated. A couple of different sacrificial polymers were used as the writing materials. Among these materials, the cross-linked amorphous polycarbonate (CPC-IV) was the best material for this application. The effect of cross-linking density on the performance of the material was investigated. A novel 3D thermal writing technology was developed by using cross-linked polymer as the writing material. A combined method utilizing the heated cantilever probe to pattern a polymer masking layer that can serve as a template for area selective atomic layer deposition techniques was developed. Another thermal probe nanolithography method, thermal probe top surface imaging, was also developed. In this method, the heated AFM tip was used to generate functional groups on the polymer surface, and ALD was used to selectively deposit TiO2 on the surface where contains those functional groups. A new poly (hydroxyl styrene) based copolymer was developed for this method. We also investigated self assembly monolayers (SAMs) as the thermal writing material. Two different SAMs were investigated. One the APTES and the other one is THP-MPTES. We demonstrated that the APTES can be patterned using thermal AFM probe, and other materials can be selectively deposited on the patterned APTES SAMs. Thermal AFM probe was used to selectively generate thiol groups from THP-MPTES SAMs, and then use these thiol groups to guide the deposition AuNPs. Some simulation and modeling works were also done to further understand these processes. FemLab was used to analyze the heat transfer in the thermal cantilever and between the heated tip and substrate. Based on kinetics of polymer thermal decomposition, we built a simple model for the selective thermal decomposition nanolithography. The experimental results can be very well fitted by this model.

Raman microscopy (RM) is a vibrational spectroscopic technique capable of obtaining sensitive measurements of molecular composition, structure, and dynamics from a very small sample volume (~1 µm). In this work, a RM system was developed for future applications in cellular radiobiology, the study of the effects of ionizing radiation on cells and tissues, with particular emphasis on the capability to investigate the internal molecular composition of single cells (10-50 µm in diameter). The performance of the RM system was evaluated by imaging 5 µm diameter polystyrene microbeads dispersed on a silicon substrate. This analysis has shown that RM of single cells is optimized for this system when using a 100x microscope objective and a 50 µm confocal collection aperture. Quantitative measurements of the spatial, confocal, and spectral resolution of the RM system have been obtained using metal nanostructures deposited on a flat silicon substrate. Furthermore, a spectral investigation of several substrate materials was successful in identifying low-fluorescence quartz as a suitable substrate for RM analysis of single cells. Protocols have been developed for culturing and preparing two human tumor cell lines, A549 (lung) and DU145 (prostate), for RM analysis, and a spectroscopic study of these two cell lines was performed. Spectra obtained from within cell nuclei yielded detectable Raman signatures from all four types of biomolecules found in a human cell: proteins, lipids, carbohydrates, and nucleic acids. Furthermore, Raman profiles and 2D maps of protein and DNA distributions within single cells have been obtained with micron-scale spatial resolution. It was also found that the intensity of Raman scattering is highly dependent on the concentration of dense nuclear material at the point of Raman collection. RM shows promise for studying the interactions of ionizing radiation with single cells, and this work has been successful in providing a foundation for the development of future radiobiological RM experiments.

Actuellement, les recherches sur la fonctionnalisation des surfaces sont en pleine effervescence. Dans ce manuscrit, nous proposons une approche innovante pour mesurer l’efficacité de cette fonctionnalisation. Cette approche est basée sur l’utilisation d’un microscope à force atomique, opérant dans un mode dit de « modulation de fréquence ». Cet outil couplé aux pointes greffées d’un nanotube de carbone, que nous appellerons « sonde », permet d’obtenir des mesures qu’il serait impossible d’effectuer avec des pointes standards. En métrologie, afin d’assurer une bonne reproductibilité des mesures, nous avons besoin d’avoir des sondes ayant des caractéristiques les plus similaires possibles. Ceci a nécessité la mise en oeuvre d’une méthode pour optimiser la fabrication des sondes, ainsi qu’une définition de critères pour les classer dans différents grades de qualités. L’incertitude de répétabilité et de reproductibilité des mesures effectuées avec des sondes de grade « A » a été quantifiée. Ces mesures ont démontré que ces sondes sont compatibles en termes de robustesse et de sensibilité pour la caractérisation de surfaces fonctionnalisées, dont l’épaisseur est supérieure à la monocouche. Des mesures de cartographie effectuées sur de deux types de surfaces fonctionnalisées ont permis de dissocier la mesure de topographie de la réponse mécanique du nanotube en interaction avec la surface. / At present, the researches on the surface functionalization are in full effervescence. In this manuscript, we propose an innovative approach to measure the efficiency of this functionalization. This approach is based on the use of an atomic force microscope, operating in a mode called "frequency modulation". This tool coupled with the grafted tips with a carbon nanotube, which we will call "probe", allows to obtain measurements which would be impossible to make with standard tips. However, in metrology, in order to ensure good reproducibility of the measurements, we need to have probes with characteristics that are as similar as possible. This required the implementation of a method to optimize the manufacture of the probes, as well as a definition of criteria to classify them in different grades of qualities. The uncertainty of repeatability and reproducibility of the measures made with probes of rank "A" were quantified. These measurements have demonstrated that these probes are compatible in terms of robustness and sensitivity for the characterization of functionalized surfaces, whose thickness is superior to the monolayer. Mapping measurements carried out on two types of functionalized surfaces enable to dissociate the topography measurement from the mechanical response of the nanotube in interaction with the surface.

Mobility of pigment-protein complexes (phycobilizomes and photosystem II playing a key role in photosynthesis) was studied by FRAP method (Fluorescence Recovery After Photobleaching). FRAP represents a fluorescence based microscopy method enabling measurement of protein mobility in living systems. The protein complexes are bleached by a laser pulse. And mobility of unbleached proteins is measured as a fluorescence recovery in the bleached area. Currently we have only limited knowledge about the mobility of photosynthetic proteins. This work was aimed at optimization of the photosynthetic protein mobility measurement by FRAP. I have performed several methodological experiments which led to the successful assessment of phycobilisome and chlorophyll-containing proteins diffusion coefficients in selected red algae (Porfyridium cruentum, Cyanidium caldarium) and cyanobacteria (Synechocystis PCC6803, Acaryochloris marina). The methodology developed and validated in my thesis was then applied in further research projects.

The main aim of my thesis was to come up with a manual for microscopic practices for elementary school's biology classes that are useful and easy to understand. The practices were drawn from ordinary textbooks (such as Fortuna, Fraus, Jinan, Natura, Nová Škola, Prodos, Scientia, SPN) that are often used for teaching pupils from 6th to 9th grade of elementary school. I also added interesting facts and practices which were drawn from the internet. I also focused on the pupil's attitude to microscoping using special questionnaires. Pupils wrote that their relationship with microscoping is positive and it helps them to understand the biology curriculum more. Furthermore, I gained information about the frequency that they practice microscoping at school. Although there are not enough biology lessons overall, I find out that most teachers usually manage to practice microscoping in class once every two weeks. In the theoretical part of the thesis, I focused on the structure of the optical microscope, which is used in classes and on the making of a temporary and permanent preparation. I also clarified the importance of microscoping practices and the pupil's opinions about it. Lastly, I added an overview of biology textbooks used in primary schools and grammar schools. Keywords: microscope, laboratory...