O chumbo é um metal muito tóxico e que tem ampla distribuição pela sua grande utilidade. Mesmo a exposição a baixos níveis (que não causam sintomas de intoxicação aguda) de chumbo é associada a desordens cognitivas e neurológicas. O osso é considerado o melhor marcador de exposição a chumbo. No tecido ósseo, vários elementos estão presentes, e alguns deles poderiam ter sua concentração modificada pela exposição a chumbo. Testou-se a hipótese de que haja variações nos elementos Zn e Mg nas tíbias de animais expostos a chumbo (por técnica de química analítica convencional). Testou-se também se variações em microelementos poderiam ser detectadas por técnicas semiquantitativas nas tíbias de controles e animais expostos a chumbo. O objetivo deste projeto foi caracterizar em animais expostos ao chumbo desde a gestação e controles as concentrações deste elemento no osso (tíbia) de animais de 28 e 60 dias. Além do chumbo, investigamos também a concentração de 2 outros microelementos no osso: zinco e magnésio. As medidas quantitativas de chumbo foram feitas no sangue total coletado ao final dos períodos. As medidas quantitativas de chumbo, cálcio, zinco e magnésio foram realizadas a partir de soluções ácidas obtidas pela dissolução total do osso, sendo estas utilizadas para quantificar por Espectrometria de Absorção Atômica no modo chama ou com Forno e Grafite. Mandíbulas foram análisadas em Microscopia Eletrônica de Varredura e utilizando os acessórios Eletron Dispersive Spectroscopy para os elementos mais abundantes e Wavelenght dispersive Spectroscopy para investigar por análise semiquantitativa os mais abundantes elementos inorgânicos no osso destes animais, como forma de verificar se há variações nestes microelementos nos animais expostos a chumbo em comparação com controles. Como o chumbo se concentra na superfície do esmalte, também analisou-se o esmalte de primeiros molares inferiores, a fim de verificar se era visto o sinal do elemento chumbo nestes dentes. Foram usados ratos Wistar machos de 28 e 60 dias divididos em 2 grupos: C 28D (n=5) e Pb 28D (n=5) e, C 60D (n=12) e Pb 60D (n=17). Em todas as medidas de chumbo dos grupos controle as concentrações obtidas foram próximas ao limite de detecção, e houve sempre diferença estatisticamente significante (P<0.0001) quando os controles foram comparados aos grupos expostos a chumbo. As concentrações de chumbo obtidas no sangue de animais do grupo Pb 28D foi de 8,0 ug/dl (± 1.1) e no grupo Pb 60D foi de 7,2 ug/dl (± 0.89). Houve diferença estatisticamente significante entre as massas das tíbias (aferidas em balança e também a partir da quantidade de cálcio dissolvida em solução) do grupo C 60D (0,86 g ± 0,13) e Pb 60D (0,61 g ±0,11)(p=0.0004). As concentrações de chumbo detectadas nos ossos do grupo Pb 28D foi 8,02 (± 1,12) e no grupo Pb 60D foi de 43,3ug/g (± 13,26). Concentração de zinco no osso do grupo C 60D foi 0,22 mg/g e no grupo Pb 60D foi 0,22 mg/g (p>0.05). A concentração de magnésio no osso do grupo C 60D foi de 4,89 mg/g ± 0,74 e no grupo Pb 60D foi de 4,98mg/g ±0,79 (p>0.05). Os microelementos mais abundantes detectados nas tíbias contralaterais por Microscopia Eletrônica de varredura por EDS/WDS foram cálcio, fósforo, carbono, oxigênio, magnésio, sódio e potássio, não tendo sido detectadas variações nestes elementos entre os grupos controle e expostos a chumbo de 60 dias. O chumbo foi detectado apenas na região cervical dos dentes molares por MEV-EDS, e, nesta posição, apenas na forma de óxido de chumbo (deposição extrínseca) que pode ser útil para fins forenses ou de determinação de contaminação desconhecida. As conclusões deste estudo são: Houve diferença estatisticamente significante entre as massas de osso do grupo Pb 60D e C 60D. Mesmo não havendo diferença entre as concentrações de chumbo no sangue total de animais de 28 e 60 dias, houve quantidade de chumbo 5 vezes maior nos animais Pb 60D. Não houve diferenças nas concentrações de zinco e magnésio entre controles e animais expostos a chumbo. Os microelementos mais abundantes detectados nas tibias contralaterais por Microscopia Eletrônica de varredura por EDS/WDS foram cálcio, fósforo, carbono, oxigênio, magnésio, sódio e potássio, não tendo sido detectadas variações semi-quantitativas nestes elementos. O chumbo não foi detectado nem nos grupos controle ou expostos por MEV, e nos molares só foi detectada no grupo exposto como uma deposição extrínseca. / Lead is a highly toxic metal that is ubiquitous do to its great usefulness. Exposure to even low levels of lead (which do not cause symptoms of acute poisoning) is associated with cognitive and neurological disorders. Bone is considered the best marker of exposure to lead. Several chemical elements are found in bone, and some of them may have a different concentration or distribution due to exposure to lead. We tested the hypothesis that there are variations in the elements zinc and magnesium in the tibia of animals exposed to lead (being those elements determined by a conventional analytic chemistry technique). It was also tested whether variations in microelements could be detected by semiquantitative microanalysis in tibias of controls and animals exposed to lead. The objective of this project was to determine the concentration of lead in bone (tibia) of animals exposed to lead from pregnancy to day 28 and day 60. Besides lead, zinc and magnesium were also determined. Lead was also determined in whole blood collected at the end of the 2 time points. Quantitative measurements of lead, calcium, zinc and magnesium have been made in solutions obtained by dissolution of the bone by Atomic Absorption Spectrometry using flame or Graphite Furnace. Tibias were analyzed by scanning electron microscopy using the accessories Electron Dispersive Spectroscopy and Wavelength dispersive Spectroscopy for semiquantitative analysis of inorganic elements in the bone and molars of these animals. Lower molars were also analyzed by these means. Male Wistar rats aged 28 and 60 days were used in these study. In both these ages there were animals in the control (C 28D, n = 5 and C 60D, n=12) and lead exposed group (Pb 28D, n = 5 and Pb 60D, n = 17). Lead concentrations found in control groups were close to the detection limit, and there was always statistically significant differences (P <0.0001) when control groups were compared with those exposed to lead. Blood lead of groups Pb 28D and Pb 60D was 8.0 ug/dl (± 1.1) and 7.2 ug/dl (± 0.89), respectively. There was a statistically significant difference between the masses of the tibia (measured in balance and also from the amount of calcium dissolved in solution): C 60D showing 0.86 g (± 0.13 g) and 0.61 g (± 0, 11) of the Pb 60D group (p = 0.0004). Lead concentrations increased five times when the Pb 28D group was compared with the Pb 60D (8.02 and 43.3 ug/g)(p<0.0001). Zinc concentrations were not different in the C 60D (0.22 mg/g) and in the Pb 60D group (0.22 mg/g)(p>0.05). Magnesium concentrations were 4.89 mg/g in the C 60D and 4.98 mg/g in the Pb 60D (p>0.05). The most abundant trace elements detected in the contralateral tibiae by SEM-EDS/WDS were calcium, phosphorus, carbon, oxygen, magnesium, sodium and potassium and no variations in these elements were detected between the control and lead exposed groups. Lead was detected only in the cervical region of molar teeth of the lead exposed groups by SEM-EDS. This finding revealed lead in the lead oxide (extrinsic deposition) which may be useful for forensic purposes or for determining unknown contaminations. Conclusions: There was a statistically significant difference between the bone masses of the Pb 60D and C 60D groups. While there is no difference between lead concentrations in whole blood of animals 28 and 60 days, the amount of lead was 5 times higher in animals Pb 60D. There were no differences in the concentrations of zinc and magnesium between controls and lead exposed animals. The most abundant trace elements detected in the contralateral tibiae by SEM-EDS/WDS were calcium, phosphorus, carbon, oxygen, magnesium, sodium and potassium, and no semi-quantitative variations in these elements were detected in relation to exposure to lead. Lead was not detected either in control or exposed groups by SEM, and in molars it was only detected in the exposed group as an extrinsic deposition.

This work deals with the evaluating of the signal level value from the sample in the environmental SEM. In work there were processed the comparison of the osciloscopic method for the evaluating of the signal level value in the environmental SEM, the method for the evaluating of the signal level from the grey level of the sample images and the method for the evaluating of the signal level from the osciloscop. There are described the advantages and disadvantages of the methods and procedures for processing the methods.

Cette thèse étudie le problème de nano-positionnement à l'intérieur d'un microscope électronique à balayage (MEB). Pour obtenir des informations de position avec rapidité et précision, une installation dédiée composée d’un vibromètre placé à l'intérieur du MEB a été mise en œuvre. Cette approche diffère de méthodes basées sur le traitement d'images, car elle permet de saisir des données en temps réel sur le comportement dynamique des structures étudiées. Dans une première étude, les perturbations mécaniques agissant à l'intérieur de la chambre à vide du microscope ont été caractérisées et leurs sources ont été identifiées. Cela a démontré comment les vibrations mécaniques externes et les bruits acoustiques peuvent influer largement sur les composants à l'intérieur du MEB par couplage mécanique, limitant ainsi la précision des manipulateurs. Dans un deuxième temps, une micro-pince du commerce a été étudiée. Une différence entre ses comportements dans l'air et dans le vide a été mise en évidence, ce qui a permis d'obtenir deux modèles dynamiques pour cet organe terminal, un pour chaque environnement. Deux lois de commande ont été proposées (commande H-infini et commande basée sur un observateur d'état étendu), afin d'obtenir en temps réel un positionnement précis dans le vide, et d'atténuer les effets des perturbations mécaniques externes. Les résultats ont été validés en simulation et expérimentalement. / This work studies the nano-positioning problem inside the scanning electron microscope (SEM). To acquire fast and accurate positional information, a dedicated setup was implemented consisting of a vibrometer placed inside the SEM. This approach differs from methods based on image processing, as it allows to capture real-time data on the dynamic behavior of structures. In a first study, the mechanical disturbances acting inside the microscope’s vacuum chamber were characterized and its sources were identified. This demonstrated how external mechanical vibrations and acoustic noises can largely influence the components inside the SEM through mechanical coupling, limiting the effective positioning precision of manipulators. Next, a commercial micro-gripper was studied, both in air and in vacuum, and the differences between its response were highlighted. This allowed to obtain two dynamic models for this end-effector, one for each environment. Two control laws were proposed (H-infinity control and Extended State Observer based control) for the system, to obtain a real-time, precise positioning in the vacuum environment and to attenuate the effects of the external mechanical disturbances. Results were demonstrated through simulation and experimental validation.