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Studying cellulose nanostructure through fluorescence labeling and advanced microscopy techniquesBabi, Mouhanad January 2022 (has links)
As the major component of the plant cell wall, cellulose is produced by all plant species at an
annual rate of over a hundred billion tonnes, making it the most abundant biopolymer on
Earth. The hierarchical assembly of cellulose glucan chains into crystalline fibrils, bundles
and higher-order networks endows cellulose with its high mechanical strength, but makes it
challenging to breakdown and produce cellulose-based nanomaterials and renewable
biofuels. In order to fully leverage the potential of cellulose as a sustainable resource, it is
important to study the supramolecular structure and hydrolysis of this biomaterial from the
nano- to the microscale.
In this thesis, we develop new chemical strategies for fluorescently labeling cellulose
and employ advanced imaging techniques to study its supramolecular structure at the singlefibril
level. The developed labeling method provides a simple and efficient route for
fluorescently tagging cellulose nanomaterials with commercially available dyes, yielding
high degrees of labeling without altering the native properties of the nanocelluloses. This
allowed the preparation of samples that were optimal for super-resolution fluorescence
microscopy (SRFM), which was used to provide for the first time, a direct visualization of
periodic disorder along the crystalline structure of individual cellulose fibrils. The
alternating disordered and crystalline structure observed in SFRM was corroborated with
time-lapsed acid hydrolysis experiments to propose a mechanism for the acid hydrolysis of
cellulose fibrils. To gain insight on the ultrastructural origin of these regions, we applied a
correlative super-resolution light and electron microscopy (SR-CLEM) workflow and
observed that the disordered regions were associated nanostructural defects present along
cellulose fibrils. Overall, the findings presented in this work provide significant
advancements in our understanding of the hierarchical structure and depolymerization of
cellulose, which will be useful for the development of new and efficient ways of breaking
down this polymer for the production of renewable nanomaterials and bio-based products
like biofuels and bioplastics. / Thesis / Doctor of Philosophy (PhD) / In this dissertation, we have studied in unprecedented detail the structure of cellulose – a
polymer that is found in every plant. As the main structural component of the plant cell wall,
cellulose endows trees with their strength and resilience while storing sunlight energy in its
chemical bonds. Since plant biomass represents eighty percent of all living matter on Earth,
cellulose is an abundant resource that can be used to produce sustainable and
environmentally benign nanomaterials and bioproducts, like biofuels and bioplastics. Our
ability to use cellulose as a renewable source of structural materials and energy is intimately
linked to our capacity to break apart its tight structural packing. Deconstructing cellulose
into various forms demands that we understand the multi-level organization of its structure
and the susceptible regions within it. To gain this information, in this thesis we develop new
labeling methods and apply state-of-the-art microscopy tools to directly visualize the
arrangement of cellulose fibrils at the nanoscale (comparable to 1/10,000 the width of a
human hair) and study their breakdown by acid treatment. The findings presented in this
work furthers our fundamental understanding of the natural structure of cellulose, which
has important implications on the development of industrial strategies to break down this
abundant and renewable biomaterial.
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Correlative multiscale imaging and quantification of bone ingrowth in porous Ti implantsGeng, Hua January 2017 (has links)
Additive manufactured porous titanium scaffolds have been extensively investigated for orthopaedic applications. The quantification of tissue response to the biomaterial implants is primarily achieved by analysing a two-dimensional (2D) stained histological section. More recently, three-dimensional X-ray micro-computed tomography (μCT) has become increasingly applied. Although histology is the gold standard, μCT allows non- destructive quantification of 3D tissue structures with minimal sample preparation and high contrast. A methodology to correlate information from both histology and μCT of a single sample might provide greater insights than either examining the results separately. However, this task is challenging because histology and μCT provide different types of information (stained tissue morphology vs. greyscale dependent on the X-ray absorption of material) and dimensionality (2D vs 3D). A semi-automated methodology was developed to directly quantify tissue formation and efficacy within an additive manufactured titanium implant using histology and μCT. This methodology was then extended to correlatively integrate nano-scale elemental information from nano- secondary ion mass spectroscopy (NanoSIMS). The correlative information was applied to investigate the impact of silver release on bone formation within a nano-silver coated additive manufactured implant. The correlative imaging methodology allowed for the quantification of the significant volumetric shrinkage (~15%) that occurs on histology slice preparation. It also demonstrated the importance of the location of the histological sectioning of the tissue and implant, revealing that up to 30% differences in bone ingrowth can be found along the entire length of the porous implant due to preferential bone ingrowth from the periphery to the centre. The quality and quantity of newly formed bone were found to be comparable between the uncoated and nano-silver coated Ti-implants, suggesting that the layer of silver nanoparticles on the Ti-implant does not negatively impact bone formation. Further, the newly formed bone at 2 weeks had a trabecula morphology with bone at the interface of Ti-implant as well as at a distant. This indicates that both contact (bone apposition on implant) and distance (bone ingrowth from host bone) osteogenesis were present in both types of implants. Finally, nanoscale elemental mapping showed silver was present primarily in the osseous tissue and was co-localised to sulphur suggesting that silver sulphide may have formed.
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Studies of macromolecular trafficking across Arabidopsis homograftsPaultre, Danaé Simone Genevieve January 2017 (has links)
Micrografting was used to study the restoration of symplasmic transport at the graft union and to examine the long-distance transport of macromolecules between scion and rootstock. New techniques were established, such as correlative imaging and single-cell analysis in microfluidic devices, to study graft development both in vivo and in vitro. Imaging of Arabidopsis homografts showed that a symplasmic domain develops in the callus stele whose function may be to contain the spread of auxin into the surrounding ground tissue. It was demonstrated, also, that recent reports of organelle transfer at the graft union cannot be explained by the formation of secondary plasmodesmata (PD) at the graft interface. While fused calli did not exchange organelles in vitro, large aggregates of the SIEVE-ELEMENT OCCLUSION RELATED protein fused to YFP (SEOR-YFP; 112 kDa) were unloaded from mature sieve tubes into living cells of the graft partner in vivo, suggesting that vascular remodelling may be a prerequisite for the exchange of organelles at the graft interface. Fusion proteins expressing organelle-targeting signals were found to translocate across the graft junction, unloading into cell files adjacent to the root protophloem. The phloem mobility of a given fusion protein was assessed using bioinformatic and statistical analysis of publicly available data. The size of a protein and its relative abundance in CCs both emerged as defining factors for subsequent phloem transport. The recipient tissue for phloem-unloaded macromolecules was identified as the phloem-pole pericycle (PPP). This cell layer is required to remove macromolecules from the terminus of the protophloem. Induced callose deposition at the PD that connect protophloem SEs to the PPP caused a restriction in unloading and a subsequent arrest in root growth. A non-cell autonomous protein of CC origin, NaKR1-1, is proposed to affect the unloading of macromolecules either by increasing the size exclusion limit (SEL) of PD within the PPP or by enabling a build-up in pressure at the protophloem terminus, due to SUC2 activity, thus allowing phloem unloading.
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Development and application of correlative STED and AFM to investigate neuronal cellsCurry, Nathan January 2018 (has links)
Over the past three decades in cellular neuroscience there has been a shift towards the view of the 'tripartite synapse', where, astrocytes -- as well as the pre-synapse and post-synapse -- are involved in synaptic signalling. The migration of astrocytes to form branched networks in the brain is, therefore, of great interest in understanding brain development and neuronal function. Migration is a complex interplay between cytoskeletal reorganisation and cell mechanical stiffness. In order to improve understanding of this process, correlative measurements of cytoskeletal organisation and mechanical stiffness are required. To investigate astrocyte migration a technique combining atomic force microscopy (AFM) with stimulated emission depletion (STED) microscopy was developed. First a custom STED microscope was developed. To facilitate the design of this system the theoretical performance of a range of STED techniques (cw-STED, time-gated STED, pulsed STED and RESOLFT) were compared, identifying that pulsed STED theoretically has the highest photon efficiency. A pulsed STED microscope, which uses adaptive optics, was then designed, developed and characterised. The microscope was found to achieve resolutions below 50 nm. The STED microscope was combined with a commercial AFM to study live cells. Using the recently developed SiR-actin and SiR-tubulin dyes and AFM probes optimised for live cell mechanical property studies, images of the actin and tubulin cytoskeleton were correlated with AFM topography and mechanical stiffness measurements. It was found that, in astrocytes, actin contributes significantly both to astrocyte stiffness and topography. Investigations of migrating cells showed differences in actin organisation and mechanical stiffness between the basis and leading edge of migration. A further study was performed, investigating the effects of the gap-junction protein connexin30, which is expressed during the early stages of brain development, on migration. This protein was found to inhibit the actin reorganisation and mechanical stiffness changes observed in basal conditions. Overall the combination of mechanosensitive AFM measurements with advanced microscopy, such as super-resolution, on live cells is a promising approach which will enable a range of investigations, for instance when studying cell structural remodeling during brain development or tumorigenesis.
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Combinaison de la microscopie de fluorescence X et de l'imagerie X par contraste de phase pour l'imagerie clinique sub-cellulaire / combined phase and X-Ray fluorescence imaging at the sub-cellular levelKosior, Ewelina 19 February 2013 (has links)
Ce travail de thèse présente une combinaison unique d'imagerie X par contraste de phase avec la fluorescence X pour des échantillons biologiques étudiés par nanosonde par fluorescence X excitée par le rayonnement synchrotron. Les récents développements dans ce domaine ouvrent la possibilité d'une imagerie chimique quantitative à l'échelle sub-cellulaire. Ceci a été rendu possible par l'utilisation d'un outil unique qui est la station de nanoimagerie X ID22NI de l'ESRF qui permet de délivrer un faisceau sub-100 nm avec un très haut flux à haute énergie entrainant une sensibilité très haute, de l'ordre de quelques centaines d'atomes pour différents éléments (Fe, Cu, Zn…). Le couplage des informations issues de l'imagerie X par contraste de phase (masse surfacique de la cellule) et de la fluorescence X (masse surfacique des éléments chimiques) a pu être obtenu pour la première fois donnant accès à une cartographie des éléments chimiques constituant les cellules et de leurs fractions massiques absolues associées. Dans l'immédiat, il n'a été possible d'étudier des cellules qui ont été congelées rapidement puis lyophilisées, cependant, une nouvelle ligne de nanoimagerie, NINA, en construction à l'ESRF, fonctionnera comme un cryomicroscope et permettra l'analyse 2D/3D d'échantillons biologiques ou non congelés hydratés. L'extension de l'imagerie chimique 2D présentée dans ce travail à une imagerie 3D représente une importante avancée pour bon nombre de problématiques scientifiques en biologie. Une des limitations de ce type d'analyse est celle des dommages radio-induits à la suite de l'irradiation de l'échantillon par un haut flux de particules ionisantes. Il existe que peu ou pas d'étude sur les effets de la nanoanalyse par fluorescence X sur les cellules lyophilisées. Nous avons combiné l'imagerie de phase à l'imagerie par fluorescence X ce qui nous permis de conclure à une rétractation des structures cellulaires accompagnée d'une volatilisation des éléments du fait de l'irradiation lors de l'analyse par fluorescence X. Ces aspects ont été confortés par des analyses utilisant une technique complémentaire non-synchrotron de microscopie ionique en transmission et à balayage (STIM). Plus important encore, nous apportons ainsi un outil rapide et non-destructif pour la cellule (imagerie X de phase) qui permet de corriger la perte de masse due à la volatilisation d'éléments légers (C, H, O, N) de la matrice cellulaire. Cette démarche permet de fiabiliser l'analyse quantitative de la composition chimique cellulaire. Cette approche sera précieuse pour corriger ces effets de perte de masse lors de futures analyses tomographiques de cellules entières congelées hydratées. Nous avons également contribué à l'étude de distribution intracellulaire de nouvelles nanoparticules d'or ou de platine fonctionnalisées. Nous avons pu exploiter les données issues de la fluorescence X pour estimer le nombre de nanoparticules et la taille des clusters internalisés au sein des cellules. Toutefois, des expériences dédiées pour des analyses sur un plus grand nombre de cellules auxquelles l'imagerie X par contraste de phase serait menée en parallèle permettraient surement de préciser plus finement ces aspects quantitatifs sur le nombre de nanoparticules intracellulaires. Dans l'ensemble ce travail ouvre la possibilité d'une imagerie chimique quantitative absolue sub-cellulaire en 2D ou 3D avec la perspective d'imagerie corrélative avec de nombreuses techniques complémentaires notamment la microscopie électronique à transmission pour l'ultrastructure, la microscopie de fluorescence pour la localisation de proteines d'intérêts et d'autres techniques d'analyses chimiques telles le NanoSIMS ou le nano-PIXE. / This work presents some recent developments in the field of hard X-ray imaging appliedto biomedical research. As the discipline is evolving quickly, new questions appear andthe list of needs becomes bigger. Some of them are dealt with in this manuscript.It has been shown that the ID22NI beamline of the ESRF can serve as a proper experimentalsetup to investigate diverse aspects of cellular research. Together with its highspatial resolution, high flux and high energy range the experimental setup providesbigger field of view, is less sensitive to radiation damages (while taking phase contrastimages) and suits well chemical analysis with emphasis on endegeneous metals (Zn, Fe,Mn) but also with a possibility for for exogoneous one’s like these found in nanoparticles(Au, Pt, Ag) study.Two synchrotron-based imaging techniques, fluorescence and phase contrast imagingwere used in this research project. They were correlated with each other on a numberof biological cases, from bacteria E.coli to various cells (HEK 293, PC12, MRC5VA,red blood cells).The explorations made in the chapter 5 allowed preparation of more establishedand detailed analysis, described in the next chapter where both techniques, X-ray fluorescenceand phase contrast imaging, were exploited in order to access absolute metalprojected mass fraction in a whole cell. The final image presents for the first timetrue quantitative information at the sub-cellular level, not biased by the cell thickness.Thus for the first time a fluorescence map serves as a complete quantitative image of acell without any risk of misinterpretation. Once both maps are divided by each otherpixel by pixel (fluorescence map divided by the phase map) they present a completeand final result of the metal (Zn in this work) projected mass fraction in ppm of dryweight. For the purpose of this calculation the analysis was extended to calibration(non-biological) samples. Polystyrene spheres of a known diameter and known densityworked very well here and allowed validation of the presented method. Different images(phase map, AFM, STIM) and profiles were compared and statement on the high accuracyof phase contrast imaging for the thickness/structures determination was made.The result on true metal projected mass fraction represents a first step to an absolutesub-cellular analysis and certainly can be improved to even closer reflect on reality.All the measurements were taken on freeze-dried cells. Thus the result is in ppm ofdry weight. In fact the measurement would have even deeper meaning if it was madeon hydrated cells. For the moment this is not possible with the existing setup of theID22NI beamline but will be possible in the future with a new beamline devoted tonano science - NINA (Nano-Imaging and Nano-Analysis). The new beamline will befurnished with a cryostage and X-ray imaging will be made on frozen-hydrated samples.Nevertheless the analysis presented in this manuscript is of undeniable importance toboth the biomedical community and to the ESRF team engaged in the NINA development.To answer the problems of cell irradiation both imaging techniques were exploitedagain. Repeating the phase contrast imaging after the fluorescence scanning allowedto show the changes induced by radiation damage during X-ray fluorescence scan. Thechanges were not only clearly visible but could be as well quantified. Together with thenumerical evaluation of damages, the dose delivered to a cell during the experiment was calculated as well. To complete the picture, a different non synchrotron-basedimaging technique, STIM, was used and compared. It is the first time that phase contrastimaging is used to monitor radiation damage effects during X-ray fluorescencemicroscopy experiments.
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Quantitative Imaging in Scanning Electron Microscope / Quantitative Imaging in Scanning Electron MicroscopeSkoupý, Radim January 2020 (has links)
Tato práce se zabývá možnostmi kvantitativního zobrazování ve skenovacím (transmisním) elektronovém mikroskopu (S|T|EM) společně s jejich korelativní aplikací. Práce začíná popisem metody kvantitativního STEM (qSTEM), kde lze stanovenou lokální tloušťku vzorku dát do spojitosti s ozářenou dávkou, a vytvořit tak studii úbytku hmoty. Tato metoda byla použita při studiu ultratenkých řezů zalévací epoxidové pryskyřice za různých podmínek (stáří, teplota, kontrastování, čištění pomocí plazmy, pokrytí uhlíkem, proud ve svazku). V rámci této části jsou diskutovány a demonstrovány možnosti kalibračního procesu detektoru, nezbytné pozadí Monte Carlo simulací elektronového rozptylu a dosažitelná přesnost metody. Metoda je pak rozšířena pro použití detektoru zpětně odražených elektronů (BSE), kde byla postulována, vyvinuta a testována nová kalibrační technika založená na odrazu primárního svazku na elektronovém zrcadle. Testovací vzorky byly různě tenké vrstvy v tloušťkách mezi 1 až 25 nm. Použití detektoru BSE přináší možnost měřit tloušťku nejen elektronově průhledných vzorků jako v případě qSTEM, ale také tenkých vrstev na substrátech - qBSE. Obě výše uvedené metody (qSTEM a qBSE) jsou založeny na intenzitě zaznamenaného obrazu, a to přináší komplikaci, protože vyžadují správnou kalibraci detektoru, kde jen malý posun úrovně základního signálu způsobí významnou změnu výsledků. Tato nedostatečnost byla překonána v případě qSTEM použitím nejpravděpodobnějšího úhlu rozptylu (zachyceného pixelovaným STEM detektorem), namísto integrální intenzity obrazu zachycené prstencovým segmentem detektoru STEM. Výhodou této metody je její použitelnost i na data, která nebyla předem zamýšlena pro využití qSTEM, protože pro aplikaci metody nejsou potřeba žádné zvláštní předchozí kroky. Nevýhodou je omezený rozsah detekovatelných tlouštěk vzorku způsobený absencí píku v závislosti signálu na úhlu rozptylu. Obecně platí, že oblast s malou tloušťkou je neměřitelná stejně tak jako tloušťka příliš silná (použitelný rozsah je pro latex 185 - 1 000 nm; rozsah je daný geometrií detekce a velikostí pixelů). Navíc jsou v práci prezentovány korelativní aplikace konvenčních a komerčně dostupných kvantitativních technik katodoluminiscence (CL) a rentgenové energiově disperzní spektroskopie (EDX) spolu s vysokorozlišovacími obrazy vytvořenými pomocí sekundárních a prošlých elektronů.
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