In this work, a characterization and optimization of the grating-based x-ray imaging technique is presented. The investigations are introduced by analytical considerations, are underpinned with numerical simulations and validated using exemplary experiments. A detailed examination of the image formation in a grating interferometer is given, highlighting the dependence of the measured signal on the profile of the gratings. Subsequently, it is shown analytically and in experiments that grating-based imaging can be performed using three basic grating arrangements, which differ in their requirements on grating fabrication and experimental implementation. By a characterization of the measurement signal for each arrangement, a dependence of the signal strength on the sample position within the interferometer is identified. The consecutive evaluation of the impact of this position dependence on radiographic and tomographic data leads to the derivation of optimized reconstruction algorithms and to a correction of resulting image artifacts. Additionally, it is shown that the simultaneous measurement of attenuation and phase images allows the determination of the atomic number of the sample, opening new possibilities for material discrimination. Apart from these investigations on the contrast formation, various imperfections of the technique are investigated: The properties of the image noise are examined in a detailed statistical analysis, yielding a fundamental understanding of the signal-to-noise behavior of the three available contrast channels. Additionally, beam-hardening artifacts at polychromatic x-ray sources are investigated and their correction by a linearization approach is resented. By a subsequent analysis of the influence of various different grating imperfections on the image quality, tolerance limits for grating fabrication are specified. Furthermore, analytical considerations show that gratings with a duty cycle of 1/3 are advantageous with respect to the signal-to-noise ratio in comparison to common gratings with a duty cycle of 1/2. In conclusion, the results, concepts and methods developed in this work broaden the understanding of grating-based x-ray imaging and constitute a step forward towards the practical implementations of the technique in imaging applications.

In patients with aortic stenosis, accurate assessment of severity with echocardiography is central to surgical decision making. But, when image quality is poor or equivocal results obtained, another robust non-invasive technique would be invaluable. Cardiac magnetic resonance (CMR) may be a useful alternative. Phase contrast CMR can measure ow and velocity, therefore it is theoretically possible to estimate the main determinant of severity aortic valve area, using the continuity approach. However, it was found that the phase contrast estimate of stroke volume, sampled in the stenotic jet, systematically underestimated left ventricular stroke volume. This underestimation was greater with increasing aortic stenosis severity. Critical clinical treatment decisions depend on the ability to reliably differentiate between patients with moderate and severe aortic stenosis. To achieve accurate estimation of aortic valve areas the velocity and ow data obtained in these turbulent, high velocity jets must be accurate. In this thesis, non-stenotic and stenotic phantoms were designed and constructed to experimentally interrogate the error. It was determined that signal loss, due to intravoxel dephasing, decreased the reliability of the measured forward ow jet velocities. Extreme signal loss in the jet eventuated in salt and pepper noise, which, with a mean velocity of zero, resulted in the underestimation. Intravoxel dephasing signal loss due to higher order motions, turbulence and spin mixing could all be mitigated by reducing the duration of the velocity sensitivity gradients and shortening the overall echo time (TE). However, improvements in an optimised PC sequence (TE 1:5ms) were not satisfactory. Flow estimates remained variable and were underestimated beyond the aortic valve. To reduce the TE further, a new phase contrast pulse sequence based on an ultrashort TE readout trajectory and velocity dependent slice excitation with gradient inversion was designed and implemented. The new sequence's TE is approximately 25% (0:65ms) of what is currently clinically available (TE 2:8ms). Good agreement in the phantom was maintained up to very high ow rates with improved signal characteristics shown in-vivo. This new phase contrast pulse sequence is worthy of further investigation as an accurate evaluation of patients with aortic stenosis. / This work in this thesis was conducted at The Auckland Bioengineering Institute, The Centre for Advanced MRI and The Oxford Centre for Clinical Magnetic Resonance in collaboration with Siemens Health care.

In patients with aortic stenosis, accurate assessment of severity with echocardiography is central to surgical decision making. But, when image quality is poor or equivocal results obtained, another robust non-invasive technique would be invaluable. Cardiac magnetic resonance (CMR) may be a useful alternative. Phase contrast CMR can measure ow and velocity, therefore it is theoretically possible to estimate the main determinant of severity aortic valve area, using the continuity approach. However, it was found that the phase contrast estimate of stroke volume, sampled in the stenotic jet, systematically underestimated left ventricular stroke volume. This underestimation was greater with increasing aortic stenosis severity. Critical clinical treatment decisions depend on the ability to reliably differentiate between patients with moderate and severe aortic stenosis. To achieve accurate estimation of aortic valve areas the velocity and ow data obtained in these turbulent, high velocity jets must be accurate. In this thesis, non-stenotic and stenotic phantoms were designed and constructed to experimentally interrogate the error. It was determined that signal loss, due to intravoxel dephasing, decreased the reliability of the measured forward ow jet velocities. Extreme signal loss in the jet eventuated in salt and pepper noise, which, with a mean velocity of zero, resulted in the underestimation. Intravoxel dephasing signal loss due to higher order motions, turbulence and spin mixing could all be mitigated by reducing the duration of the velocity sensitivity gradients and shortening the overall echo time (TE). However, improvements in an optimised PC sequence (TE 1:5ms) were not satisfactory. Flow estimates remained variable and were underestimated beyond the aortic valve. To reduce the TE further, a new phase contrast pulse sequence based on an ultrashort TE readout trajectory and velocity dependent slice excitation with gradient inversion was designed and implemented. The new sequence's TE is approximately 25% (0:65ms) of what is currently clinically available (TE 2:8ms). Good agreement in the phantom was maintained up to very high ow rates with improved signal characteristics shown in-vivo. This new phase contrast pulse sequence is worthy of further investigation as an accurate evaluation of patients with aortic stenosis. / This work in this thesis was conducted at The Auckland Bioengineering Institute, The Centre for Advanced MRI and The Oxford Centre for Clinical Magnetic Resonance in collaboration with Siemens Health care.

In patients with aortic stenosis, accurate assessment of severity with echocardiography is central to surgical decision making. But, when image quality is poor or equivocal results obtained, another robust non-invasive technique would be invaluable. Cardiac magnetic resonance (CMR) may be a useful alternative. Phase contrast CMR can measure ow and velocity, therefore it is theoretically possible to estimate the main determinant of severity aortic valve area, using the continuity approach. However, it was found that the phase contrast estimate of stroke volume, sampled in the stenotic jet, systematically underestimated left ventricular stroke volume. This underestimation was greater with increasing aortic stenosis severity. Critical clinical treatment decisions depend on the ability to reliably differentiate between patients with moderate and severe aortic stenosis. To achieve accurate estimation of aortic valve areas the velocity and ow data obtained in these turbulent, high velocity jets must be accurate. In this thesis, non-stenotic and stenotic phantoms were designed and constructed to experimentally interrogate the error. It was determined that signal loss, due to intravoxel dephasing, decreased the reliability of the measured forward ow jet velocities. Extreme signal loss in the jet eventuated in salt and pepper noise, which, with a mean velocity of zero, resulted in the underestimation. Intravoxel dephasing signal loss due to higher order motions, turbulence and spin mixing could all be mitigated by reducing the duration of the velocity sensitivity gradients and shortening the overall echo time (TE). However, improvements in an optimised PC sequence (TE 1:5ms) were not satisfactory. Flow estimates remained variable and were underestimated beyond the aortic valve. To reduce the TE further, a new phase contrast pulse sequence based on an ultrashort TE readout trajectory and velocity dependent slice excitation with gradient inversion was designed and implemented. The new sequence's TE is approximately 25% (0:65ms) of what is currently clinically available (TE 2:8ms). Good agreement in the phantom was maintained up to very high ow rates with improved signal characteristics shown in-vivo. This new phase contrast pulse sequence is worthy of further investigation as an accurate evaluation of patients with aortic stenosis. / This work in this thesis was conducted at The Auckland Bioengineering Institute, The Centre for Advanced MRI and The Oxford Centre for Clinical Magnetic Resonance in collaboration with Siemens Health care.

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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No. of bitstreams: 1 maximino_md_me_prud.pdf: 5933036 bytes, checksum: 96040f746ca01578e5036f01f3d89e6e (MD5) Previous issue date: 2018-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho trata do estudo da interação entre os poluentes emergentes amoxicilina (AMX) e azul de metileno (AM) com modelos-simples de membrana biológica compostas de fosfolipídios zwiteriônicos (DPPC e DOPC), através de filmes de Langmuir e vesículas unilamelares gigantes (GUVs). As membranas foram expostas aos poluentes de forma individual e também misturados (MIX) para que se aproximasse de uma situação real, neste caso, o chamado efeito coquetel, no qual os poluentes são encontrados misturados no ambiente. O objetivo principal foi investigar as possíveis interações entre os poluentes e os fosfolipídios e os consequentes efeitos nas membranas. Nos filmes de Langmuir, as isotermas π-A (pressão superficial por área molecular média) de DPPC revelaram que a AMX e o AM são expulsos da interface ar-água conforme a fase condensada é atingida. O DOPC demonstrou ser menos afetado pela AMX, uma vez que a isoterma não apresentou deslocamento. Contudo, o DOPC se mostrou mais susceptível a interagir com o AM, visto que um deslocamento significativo foi observado. Para ambos os lipídios a MIX causou maiores efeitos, evidenciados por deslocamentos maiores nas isotermas. Análises de módulo compressional mostraram um pequeno aumento na fluidez do filme de DPPC, não observado para o DOPC. As medidas de microscopia de ângulo de Brewster (BAM) na presença dos poluentes revelaram mudanças na morfologia dos filmes, afetando a formação dos domínios de DPPC e impedindo a formação dos mesmos nas monocamadas de DOPC. Os espectros de PMIRRAS para o DPPC indicaram possíveis interações eletrostáticas dos poluentes com a cabeça polar do lipídio, assim como uma desordem na região da sua cauda apolar. A monocamada de DOPC foi bastante afetada pois ocorreram mudanças nas vibrações de quase todos os grupos químicos do lipídio, apresentando inclusive mudanças na região da cauda do lipídio. Os espectros de emissão da sonda LAURDAN nas LUVs revelaram um aumento da rigidez para ambos os lipídios, diferentemente do observado pelas análises de elasticidade da monocamada de Langmuir. As GUVs para ambos os fosfolipídios, ao contrário das monocamadas, apresentaram maior efeito sobre os poluentes aplicados de forma individual, visto que para os poluentes avaliados individualmente foram observados a perda de contraste de fase para os lipídios na presença do AM, assim como mudanças na estabilidade para as GUVs de DOPC em AMX. Tais efeitos não foram observados para a MIX, devido as diferenças estruturais dos modelos de membrana. / The present work is about the interaction between the emerging pollutants amoxicillin (AMX) and methylene blue (MB), with biological membrane models of zwitterionic phospholipids (DPPC and DOPC), through Langmuir films and giant unilamellar vesicles (GUVs). The membrane was exposed to the presence of the pollutants individually, still being performed the analysis with the mixture (MIX) of both in order to get closer to a real situation, in which the pollutants are found mixed in the environment. The main goal was to investigate the possible interactions between the pollutants and phospholipids. The π-A (surface pressure by mean molecular area) isotherms of DPPC reveal that in the condensed phase the lipid expelled the AMX and MB of the air-water interface. The DOPC showed to be less affected by the AMX, once the isotherm did not present a displacement. However, the DOPC showed to be more susceptible to interact with the MB, since significant displacements were observed. In both lipids, the MIX showed to be more effective, causing bigger displacements. The analysis of compressional modulus showed a small increase of the fluidity of the DPPC film, which was not observed to the DOPC. The Brewster Angle Microscopy (BAM) measurements in the presence of the pollutants revealed changes in the morphology, affecting the domains formation of DPPC and inhibiting the formation of the same in monolayers. The PM-IRRAS specters to the DPPC indicated possible electrostatic interactions with the head, as well as a disorder in the tail. The DOPC monolayer was quite affected because there have been changes in almost all chemical groups of lipid, including changes in the tail region of the lipid. The emission spectrum from the dye LAURDAN in the LUVs revealed an increase in the rigidity for both lipids, different from the observed in the analysis of elasticity in the Langmuir monolayers. The GUVs for both phospholipids, unlike the monolayers, showed a major effect on the pollutants applied individually since that were observed losses of phase contrast for the lipids in the presence of MB, such as changes in the stability of the DOPC GUVs in AMX. Such effects were not observed in the MIX, due to the structural differences in the membrane models.

Orientador: Carlos Manuel Giles Antunez de Mayolo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-12T20:54:38Z (GMT). No. of bitstreams: 1 Mardegan_JoseRenatoLinares_M.pdf: 9598511 bytes, checksum: eff03242148c4fb5d74188492a5542d5 (MD5) Previous issue date: 2009 / Resuno: Esta dissertação de mestrado teve por objetivo a realização de imagens pela técnica de imagens por contraste de fase utilizando o método de imagens realçadas por difração (IRD) para analisar amostras histológicas de mama in vitro. Os sistemas para produção das IRD foram desenvolvidos utilizando um gerador convencional no Laboratório de Cristalografia Aplicada e Raios X (LCARX - UNICAMP) e também com radiação síncrotron no Laboratório Nacional de Luz Síncrotron (XRD2 - LNLS). As amostras estudadas foram corpos de provas, um inseto e tecidos de mama com carcinoma lobular invasivo e carcinoma ductal invasivo fornecidas pelo Centro de Atenção Integral a Saúde da Mulher (CAISM - UNICAMP). As imagens foram realizadas pelo método convencional e também pelo método de IRD. As imagens realçadas por difração de corpos de prova, do inseto e dos tecidos com câncer de mama mostraram resolução superior ao comparadas com imagens convencionais principalmente das interfaces entre tecidos em uma amostra, o que resulta em um diagnostico mais preciso do paciente. As imagens obtidas pelo método IRD apresentam contraste, visibilidade e razão-sinal ruído muito superiores quando comparados com as imagens obtidas pela técnica convencional de absorção. A imagem obtida do tecido com carcinoma lobular invasivo apresentou maiores detalhes ao compará-la com a imagem convencional porém não foi possível visualizar precisamente as bordas do tumor. As imagens realçadas por difração têm qualidade superior devido à utilização de um cristal analisador colocado após a amostra. Este cristal funciona como uma estreita fenda angular resultando em imagens com ganho de contraste em relação às imagens pelos métodos convencionais. Utilizando o algoritmo de tratamento das IRD foram obtidas as imagens de Absorção Aparente e Refração ajustando o cristal analisador em diferentes posições da curva de difração e não apenas na posição de meia altura da curva de refletividade. A escolha de diferentes posições da curva mostrou que as imagens de absorção aparente, refração e as imagens obtidas nestes flancos ganham em resolução quando ajustadas para posições distantes do topo. Valores calculados para os parâmetros sinal ruído borda e visibilidade em imagens obtidas em posições distantes do topo chegaram a ser cerca de dez vezes maiores que nas imagens convencionais para as amostras de corpos de prova. Isto ocorre porque o cristal analisador está selecionando diferentes feixes refratados na amostra. / Abstract: In this dissertation we have produced in vitro breast images by a phase contrast imaging technique using the diffraction enhanced imaging (DEI) method. The DEI setup was implemented both at the X-ray applied Crystallography Laboratory (LCARX) using a conventional generator and at the XRD2 beamline of the Brazilian National Synchrotron Light Laboratory (LNLS) in Campinas. Two types of breast cancer were investigated from breast tissues provided by the University Center for Women Health (CAISM-UNICAMP) through collaboration with a professional mastologist: invasive lobular carcinoma and invasive ductal carcinoma. Other images were performed on well characterized test objects and biological samples. The images obtained were compared with images obtained by the conventional absorption contrast method. The diffraction enhanced images of test objects, insect and breast tissues with cancer showed higher resolution as compared with the conventional images mainly from the boundaries of the sample. The tissue with invasive lobular carcinoma had details increased when compared with a conventional image, but it was not possible to clearly visualize the interfaces of the tumour in this case. The diffraction enhanced images had higher quality because this system uses an analyzer crystal placed after the sample. This crystal serves as a narrow angular slit which resulted in images with gain in contrast as compared to the conventional images methods. The DEI algorithm to obtain two new images so called "Apparent Absorption" and "Refraction" images were used to the breast tissue and test objects images taken at symmetrical half intensity sides of the rocking curve. Furthermore, this algorithm was also used to images taken at symmetrical positions of the analyzer further away from the top resulting in images with higher resolution and signal to noise ratio. / Mestrado / Física da Matéria Condensada / Mestre em Física

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