Mon travail de thèse, débuté en septembre 2005, a été consacré au développement de l’émergente technique d’imagerie par spectrométrie de masse ToF-SIMS. Une première partie de ce travail a été orientée vers des aspects fondamentaux avec l’utilisation d’une source d’ions fullerènes comme faisceau d’ions primaires, mais aussi comme faisceau de pulvérisation. Le but était d’éprouver la possibilité de réaliser une imagerie 3D sur des échantillons biologiques par spectrométrie de masse ToF-SIMS. Une seconde partie de mon travail de thèse a été consacrée à l’utilisation de la technique proprement dite. Différents domaines d’application ont été étudiés, comme l’archéologie, avec l’analyse de la composition des patines recouvrant les statues rituelles de la tribu Dogon (Mali) ou l’étude de la dégradation de cheveux de momies chinoises. Un troisième projet a consisté à analyser in situ les surfactines, une famille de cyclodepsipeptides amphiphiles, sur des profils de colonisation de surface de Bacillus subtilis. Cette analyse qualitative par imagerie ToF-SIMS a été complétée par une analyse quantitative par spectrométrie de masse MALDI-TOF. Enfin, le dernier projet mis en œuvre durant ma thèse concerne la recherche de biomarqueurs lipidiques de la stéatose hépatique non alcoolique chez l’humain. Les résultats ont mis en évidence la complémentarité de l’imagerie par spectrométrie de masse avec d’autres techniques permettant la localisation de composés ciblés, comme la coloration histologique. Les résultats de cette thèse démontrent que l’imagerie par spectrométrie de masse ToF-SIMS peut être appliquée dans des domaines aussi divers que l’archéologie, la microbiologie ou la médecine. / My PhD’s work has been devoted to the development of the emergent technique ToF-SIMS imaging. The first part of my work was dedicated to fondamental aspects with the use of a fullerene ion source as a primary ion beam or sputtering ion beam. We expected to realize 3D imaging. The second part of my work consisted to applications of the mass spectrometry imaging. Several application fields were studied, as archeology as with the analysis of patina of the Dogon statuary or chinese mummy hair. A third project was dedicated to the in situ biomarker research from human liver biopsies. The goal of this study was to identify a potential lipid biomarker of the non-alcoholic fatty liver disease. The last part of this manuscript is devoted to the in situ qualitative and quantitative analysis of surfactins (a family of heptacyclodepsipeptides) on a Bacillus subtilis swarming community. We combined ToF-SIMS imaging for qualitative analysis and localization of surfactins within the swarming pattern and MALDI-TOF mass spectrometry for the quantification of these species. The results ot this PhD’s work show that ToF-SIMS imaging could be applied to various fields of research as archeology, microbiology and medicine.

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Xylen är ett starkt toxiskt ämne, som trots att dess hälsofarliga egenskaper länge varit kända, fortsatt har använts som klarningsmedel i den histologiska processen. Syftet med den här studien var att undersöka om det betydligt mindre hälsofarliga lösningsmedlet, Histolab Clear, kunde substituera xylen som klarningsmedel, utan att kompromissa med kvalitén på de histologiska glas som laboratoriet lämnar ut för granskning. Två serier med samma uppsättning vävnadsbitar preparerades och dehydrerades med var sitt klarningsmedel. Klossarna granskades och bedömdes utifrån snittbarhet, färgkvalité och snittkvalité. Vid jämförelse av de två serierna visades inga betydande skillnader inom något av bedömningsområdena. Att resultatet är likvärdigt inom alla tre bedömningsområden stärker slutsatsen att Histolab Clear är ett bra alternativ som xylensubstitut. Däremot visar studien även att klossarnas snittbarhet tydligt påverkas av tjockleken på den utskurna vävnadsbiten. Tjocka vävnadsskivor ger en sämre snittbarhet vilket kan påverka snittets kvalité. För att säkra snitt av hög kvalité med välbevarad morfologi, bör det vid utskärningen inte tas vävnadsskivor som är tjockare än fyra millimeter. / Xylene is a highly toxic substance that, despite its long-known hazardous characteristics, still has been used as a clearing agent in the histological process. The purpose of this study was to investigate whether the significantly less toxic solvent, Histolab Clear, could substitute xylene as a clearing agent, without compromising the quality of the histological slides released by the laboratory for examination. Two series with the same set of tissue pieces were prepared and dehydrated with a clarifying agent each. The blocks were examined and assessed based on sectionability, quality of color and quality of section. When comparing the two series, no significant differences were shown in any of the assessment areas. The fact that the result is equivalent in all three assessment areas reinforces the conclusion that Histolab Clear is a good alternative to substitute xylene. However, the study also shows that the sectionability of the blocks is clearly affected by the thickness of the tissue. Thick tissue slices result in poorer sectionability, which can affect the quality of the slide. To ensure high quality sections, with well-preserved morphology, tissue slices that are thicker than four millimeters should not be cut during grossing.

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This dissertation presents an investigation of micromachined three-dimensional microelectrode arrays (3-D MEAs) targeted toward in-vitro and in-vivo biomedical applications. Current 3-D MEAs are predominantly silicon-based, fabricated in a planar fashion, and are assembled to achieve a true 3-D form: a technique that cannot be extended to micro-manufacturing. The integrated 3-D MEAs developed in this work are polymer-based and thus offer potential for large-scale, high volume manufacturing. Two different techniques are developed for microfabrication of these MEAs - laser micromachining of a conformally deposited polymer on a non-planar surface to create 3-D molds for metal electrodeposition; and metal transfer micromolding, where functional metal layers are transferred from one polymer to another during the process of micromolding thus eliminating the need for complex and non-repeatable 3-D lithography processes. In-vitro and in-vivo 3-D MEAs are microfabricated using these techniques and are packaged utilizing Printed Circuit Boards (PCB) or other low-cost manufacturing techniques. To demonstrate in-vitro applications, growth of 3-D co-cultures of neurons/astrocytes and tissue-slice electrophysiology with brain tissue of rat pups were implemented. To demonstrate in-vivo application, measurements of nerve conduction were implemented. Microelectrode impedance models, noise models and various process models were evaluated. The results confirmed biocompatibility of the polymers involved, acceptable impedance range and noise of the microelectrodes, and potential to improve upon an archaic clinical diagnostic application utilizing these 3-D MEAs.