Feasibility study of in vivo MRS for functional experiments

Orientador: Gabriela CastellanoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb WataghinResumo: A técnica de espectroscopia por ressonância magnética (MRS, do inglês Magnetic Resonance Spectroscopy)baseada no núcleo do hidrogênio (1 H -MRS)tem sido muito usada em estudos neurológicos na determinação de padrões metabólicos para várias patologias. A informação fornecida por esta técnica é única, pois permite monitorar níveis de metabólitos específicos, envolvidos em vários aspectos da função cerebral. Até o momento, a grande maioria dos estudos de 1 H -MRS in vivo têm sido "estáticos ", no sentido de que uma única medida é feita, sem se preocupar com informação temporal. Isso se deve à baixa razão sinal-ruído inerente a esta técnica, que obriga à realização de aquisições longas e médias temporais, sacrificando a resolução temporal. No entanto,o advento da técnica de ressonância magnética funcional e de outras técnicas de neuroimagem dinâmicas, que medem parâmetros dinâmicos como fluxo sanguíneo, taxa de oxigenação do sangue, atividades elétrica e magnética do cérebro, naturalmente fez surgir o interesse em se ter uma técnica que pudesse fornecer informação dinâmica sobre as vias metabólicas associadas à função cerebral - uma MRS funcional. O objetivo deste trabalho foi, portanto, verificar a viabilidade da utilização da técnica de MRS in vivo em experimentos funcionais, tendo sido o primeiro realizado nesta área, no Brasil. Para isso foi primeiramente realizado um estudo extensivo sobre os poucos trabalhos existentes na área e as bases bioquímicas da ativação neuronal. Em seguida, foram realizados experimentos e desenvolvidos vários métodos de análise para tentar detectar a variação temporal dos principais metabólitos presentes num espectro cerebral (N-acetil-aspartato total: NAA, Creatina total: Cre, grupo Colina: Cho, grupo Glutamato/Glutamina: Glx, e Lactato: Lac)em indivíduos saudáveis, durante um experimento com estímulo visual. Os experimentos apresentaram uma série de dificuldades, e não foi possível alcançar a alta resolução temporal desejada, sendo que a resolução média ficou na ordem de minutos -o que concorda, no entanto, com a maioria dos trabalhos encontrados na literatura. Não detectamos variações significativas nos níveis de NAA, Cre e Cho, o que também está de acordo com a maioria dos estudos encontrados na literatura. Por outro lado, foram encontradas variações nos níveis de Lac (que aumentaram com o estímulo, o que concorda com a literatura, porém não voltaram ao nível basal após o estímulo, o que não concorda), e variações nos níveis do grupo Glx (que aumentaram com o estímulo, voltando em seguida ao nível basal, o que concorda com a literatura). Embora os resultados encontrados não tenham sido totalmente concordantes com a literatura e não tenha sido possível melhorar a resolução temporal (em relação aos trabalhos da literatura), acreditamos que este trabalho deixa uma significativa contribuição através dos diversos protocolos experimentais e métodos de análise testados, e abre o caminho para pesquisas futuras na áreaAbstract: The technique of Magnetic Resonance Spectroscopy using the hydrogen nucleus (1 H -MRS) has been widely used for neurologic research for determining metabolic patterns for many pathologies. The type of information provided by this technique is unique, since it allows monitoring specific metabolic concentrations involved in cerebral function.Until now, the majority of the in vivo 1 H -MRS studies have been "static ", meaning that data acquisition is done with no concern for temporal information. The reason for this is the low signal-to-noise ratio (SNR)inherent to this technique, which imposes long acquisitions and time averaging that sacrifices temporal resolution. However, the development of function Magnetic Resonance Imaging (fMRI)and other time resolved neuroimaging techniques, that measure dynamic parameters such as blood flow, blood oxygenation rate, electric and magnetic cerebral activity, naturally brought up the interest on a technique that would allow a time resolved measure of the specific metabolic concentrations involved on cerebral metabolism - something like a functional MRS. Therefore the goal of this work was to study the feasibility of using the in vivo MRS technique for functional experiments, being the .rst of its kind performed in Brazil.To do so,initially an extensive bibliographic research was done, to comprehend not only the details of these experiments but also the neurochemistry behind the MRS signal.Next, experiments were performed and many analysis methods were developed, in order to attempt to detect temporal variations of the main metabolites present on a typical cerebral spectrum (total N-acetyl-aspartic acid: NAA, total Creatine: Cre, total Choline: Cho, Glutamine/Glutamate group: Glx and Lactate: Lac) in healthy subjects, during a visual stimulation experiment. Those experiments presented a series of difficulties and the desired high temporal resolution was not attained, achieving an average resolution of minutes - the same resolution found on the majority of the works published. We did not detect any significant variation of the NAA, Cre or Cho levels, which supports the conclusions of other works published. On the other hand, we detected variations in the Lactate levels (which increased with the stimulus, as reported in other works, but did not return to base-line levels, which disagrees with the published works), and in the Glx levels (which increased with the stimulus, returning to baseline levels after it, which agrees with the published literature). Although the results found are not totally in agreement with the published literature and it was not possible to improve the temporal resolution (compared to published works), we believe that this work leaves a significant contribution to the field, through the experimental protocols and analysis methods tested, and opens paths for future research in this areaMestradoFísicaMestre em Físic

Location of water in fresh sugarcane bagasse observed by synchrotron X-ray microtomography

Sugarcane bagasse is a vast lignocellulosic byproduct generated in the industry with ~50% humidity (1 kg dry matter associated with 1 kg water). Although the presence of water brings deleterious consequences for combustion, storage and sugar extraction, the location of water in fresh bagasse remains unknown. In this work, we use synchrotron X-ray microtomography for non-invasive 3D imaging of fresh bagasse particles, which allows the visualization of intraparticle water. The sclerified fiber cells in the sheaths surrounding xylem vessels are often found full of water. We suggest this can be juice preserved from the native stalks as many sclerified fibers seem to keep their structural integrity despite the mechanical action during sugarcane crushing. The microtomograms of fresh bagasse also shows mineral particles adhered to biomass surfaces, with adhesion presumably favored by the presence of water. In summary, this work unveils the location of water in fresh bagasse, solving an old mystery of sugarcane technology

Illuminating the Brain With X-Rays: Contributions and Future Perspectives of High-Resolution Microtomography to Neuroscience

The assessment of three-dimensional (3D) brain cytoarchitecture at a cellular resolution remains a great challenge in the field of neuroscience and constant development of imaging techniques has become crucial, particularly when it comes to offering direct and clear obtention of data from macro to nano scales. Magnetic resonance imaging (MRI) and electron or optical microscopy, although valuable, still face some issues such as the lack of contrast and extensive sample preparation protocols. In this context, x-ray microtomography (μCT) has become a promising non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens. It is a new supplemental method to be explored for deciphering the cytoarchitecture and connectivity of the brain. This review aims to bring together published works using x-ray μCT in neurobiology in order to discuss the achievements made so far and the future of this technique for neuroscience

Shape Tailored Magnetic Nanorings for Intracellular Hyperthermia Cancer Therapy

This work explores a new class of vortex/magnetite/iron oxide nanoparticles designed for magnetic hyperthermia applications. These nanoparticles, named Vortex Iron oxide Particles (VIPs), are an alternative to the traditional Superparamagnetic Iron Oxide Nanoparticles (SPIONs), since VIPs present superior heating power while fulfilling the main requirements for biomedical applications (low cytotoxicity and nonremanent state). In addition, the present work demonstrates that the synthesized VIPs also promote an internalization and aggregation of the particles inside the cell, resulting in a highly localized hyperthermia in the presence of an alternating magnetic field. Thereby, we demonstrate a new and efficient magnetic hyperthermia strategy in which a small, but well localized, concentration of VIPs can promote an intracellular hyperthermia process

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features in the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe$_3$O$_4$ core encased by a 25-nm-thick fluorescent silica (SiO$_2$) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles

A correlation analysis of Light Microscopy and X-ray MicroCT imaging methods applied to archaeological plant remains’ morphological attributes visualization

In this work, several attributes of the internal morphology of drupaceous fruits found in the archaeological site Monte Castelo (Rondonia, Brazil) are analyzed by means of two different imaging methods. The aim is to explore similarities and differences in the visualization and analytical properties of the images obtained via High Resolution Light Microscopy and X-ray micro-computed tomography (X-ray MicroCT) methods. Both provide data about the three-layered pericarp (exo-, meso- and endocarp) of the studied exemplars, defined by cell differentiation, vascularisation, cellular contents, presence of sclerenchyma cells and secretory cavities. However, it is possible to identify a series of differences between the information that can be obtained through each of the methods. These variations are related to the definition of contours and fine details of some characteristics, their spatial distribution, size attributes, optical properties and material preservation. The results obtained from both imaging methods are complementary, contributing to a more exhaustive morphological study of the plant remains. X-ray MicroCT in phase-contrast mode represents a suitable non-destructive analytic technique when sample preservation is required

Core-shell Fe@Fex_xOy_y nanoring system: A versatile platform for biomedical applications

Iron oxide (maghemite and magnetite) nanoparticles are the most commonly used magnetic materials in nanomedicine because of their high biocompatibility. However, their low saturation magnetization (60–90 emu/g) limits their applicability. Here, we report a new core–shell (Fe@Fe$_x$O$_y$) nanoring system, which combines the high magnetic saturation of a metallic iron core (220 emu/g) and the biocompatibility of an iron oxide shell. To produce these nanostructures, hematite (α-Fe$_2$O$_3$) nanorings were annealed in a H$_2$ gas atmosphere for different periods to optimize the amount of metallic iron percentage (δ) in the system. Thus, nanostructures with different magnetic saturation (97 to 178 emu/g) could be obtained; based on their metallic iron content, these particles are labeled as Vortex Iron oxide Particle δ (VIPδ). Micromagnetic simulations confirmed that the VIPδ nanorings exhibit a vortex configuration, guaranteeing low remanence and coercitivity. Moreover, the system shows good biocompatibility in various assays as determined through cell viability measurements performed using two different human cell lines, which were exposed to VIP78% for 24 h. Therefore, VIPδ nanorings combine a magnetic vortex state and biocompatibility with their high magnetic saturation and can thus serve as a platform that can be tuned during the synthesis based on desired biomedical application

High-resolution synchrotron-based X-ray microtomography as a tool to unveil the three-dimensional neuronal architecture of the brain

The assessment of neuronal number, spatial organization and connectivity is fundamental for a complete understanding of brain function. However, the evaluation of the three-dimensional (3D) brain cytoarchitecture at cellular resolution persists as a great challenge in the field of neuroscience. In this context, X-ray microtomography has shown to be a valuable non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens, arisen as a new method for deciphering the cytoarchitecture and connectivity of the brain. In this work we present a method for imaging whole neurons in the brain, combining synchrotron-based X-ray microtomography with the Golgi-Cox mercury-based impregnation protocol. In contrast to optical 3D techniques, the approach shown here does neither require tissue slicing or clearing, and allows the investigation of several cells within a 3D region of the brain

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity.

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI's quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 ± 1.0 nm and 4.0 ± 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI’s quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 ± 1.0 nm and 4.0 ± 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity