L'imagerie par spectrométrie de masse est d’un grand intérêt pour aborder les questions biologiques en fournissant simultanément des informations chimiques et spatiales. En particulier, la spectrométrie de masse baptisée TOF-SIMS est bien reconnue par sa haute résolution spatiale (< 1 μm), qui est essentielle pour révéler l'information chimique dans une zone submicronique. L'emploi croissant de cette technique dans la caractérisation des échantillons biologiques a bénéficié du développement de nouvelles sources d'ions d’agrégats. Cependant, les processus d'ionisation/désorption des analytes sous les impacts d’agrégats lourds sont encore mal compris. D'un autre côté, techniquement, les instruments TOF-SIMS commerciaux actuels ne peuvent pas fournir une résolution en masse suffisante ni une précision sur la détermination de la masse pour l'identification moléculaire, ce qui rend les analyses de systèmes biologiques complexes très difficiles, et nécessite le recours à la fragmentation MS/MS. Cette thèse vise à mieux comprendre la production d'ions sous l’impact d’agrégats lourds et à explorer la capacité MS/MS du spectromètre de masse par temps de vol combiné à l’imagerie ionique en utilisant le spectromètre de masse PHI nanoTOF II. Ce dernier point a été réalisé en cartographiant en haute résolution spatiale des métabolites importants de bois. Pour comprendre la production d'ions sous les impacts d’agrégats d'argon massifs, l'énergie interne des ions secondaires a été mesurée en utilisant la mesure du taux de survie d'une série d'ions benzylpyridinium. L'étude de diverses conditions d'impact (énergie, vitesse, taille des agrégats) a montré que la vitesse joue le rôle majeur dans la distribution d'énergie interne et la fragmentation moléculaire dans le régime à faible énergie par atome (E/n < 10 eV).Les capacités de la fragmentation MS/MS et d'imagerie en parallèle du spectromètre PHI nanoTOF II nouvellement conçu ont été évalués par cartographie MS/MS in situ des métabolites bioactifs rubrynolide et rubrenolide dans les espèces amazoniennes de bois Sextonia rubra, ainsi qu’une identification in situ des métabolites précurseurs. L'imagerie TOF-SIMS 2D et 3D a permis de localiser les cellules où cette biosynthèse s’effectue. Les résultats ont conduit à la proposition d'une voie possible de biosynthèse des deux métabolites. Pour étendre l'application de l'imagerie TOF-SIMS dans l'analyse chimique du bois, la distribution radiale des extraits de bois dans le duramen du bois du mélèze européen a également été étudiée. / Mass spectrometry imaging has been shown of great interest in addressing biological questions by providing simultaneously chemical and spatial information. Particularly, TOF-SIMS is well recognized for its high spatial resolution (< 1 µm) which is essential in disclosing chemical information within a submicron area. The increasing use of TOF-SIMS in characterizing biological samples has greatly benefited from the introduction of new cluster ion sources. However, the ionization/desorption of the analytes under impacts of large clusters is still poorly understood. On the other hand, technically, current commercial TOF-SIMS instruments generally cannot provide sufficient mass resolution or mass accuracy for molecular identification, making analyses of complex biological systems especially challenging when no MS/MS fragmentation is available. Thus this thesis is aimed to get a better understanding of ion production under cluster impacts, to explore the MS/MS capability of the parallel imaging MS/MS Spectrometer (PHI nanoTOF II), as well as to apply TOF-SIMS to map important wood metabolites with high spatial resolution.In order to understand ion production under impacts of massive argon clusters, internal energy distributions of secondary ions were measured using survival yield method which involves the analyses of a series of benzylpyridinium ions. Investigation of various impacting conditions (energy, velocity, cluster size) suggested that velocity of the clusters play a major role in internal energy distribution and molecular fragmentation in the low energy per atom regime (E/n < 10 eV). The MS/MS fragmentation and parallel imaging capabilities of the newly designed PHI nanoTOF II spectrometer were evaluated by in situ MS/MS mapping of bioactive metabolites rubrynolide and rubrenolide in Amazonia wood species Sextonia rubra. Then this parallel imaging MS/MS technique was applied to perform in situ identification of related precursor metabolites in the same tree species. 2D and 3D TOF-SIMS imaging were carried out to target the plant cells that biosynthesize rubrynolide and rubrenolide. The results led to the proposal of a possible biosynthesis pathway of these two metabolites. In addition, to expand the application of TOF-SIMS imaging in wood chemistry analysis, radial distribution of wood extractives in the heartwood of European larch was also investigated.

<p>   </p> <p>Ambient Mass Spectrometry Imaging (MSI) is a powerful analytical tool in biomedical research that enables simultaneous label-free spatial mapping of hundreds of molecules in biological samples under native conditions. Nanospray desorption electrospray ionization (nano-DESI) is an emergent ambient MSI technique developed in 2010 that uses localized liquid extraction of molecules directly from surfaces. Like other liquid-extraction based techniques, nano-DESI relies on gentle removal of molecules from surfaces and soft ionization. High sensitivity and spatial resolution, versatility of the solvent composition, which may be used to tailor the extraction and ionization of selected molecules, quantification capabilities at the single-pixel level as well as compensation for matrix effects by adding a known standard to the solvent, and online derivatization are key features of nano-DESI MSI that position it as a unique analytical tool for studying biological systems. </p> <p>Despite the advantages that nano-DESI provides, there are still challenges associated with the structural characterization, extraction, and detection of certain molecular classes. Therefore, my dissertation research has focused on addressing these analytical challenges by developing innovative approaches that substantially enhance the performance of the nano-DESI technique in the study of complex biological systems. </p> <p>In this thesis, a systematic study of the solvent composition is carried out to aid in the detection of neutral lipids such as triglycerides thereby expanding the molecular coverage of nano-DESI experiments. Taking advantage of the versatility of the solvent composition, I developed an approach for the online derivatization of unsaturated lipids into lipid hydroperoxides using the reaction of singlet oxygen with C=C bonds. This method further expands the specificity of nano-DESI MSI by enabling the detection and imaging of positional lipid isomers. To aid in the analysis of complex mixtures and provide additional structural information in the form of collision cross sections, coupling of nano-DESI with a drift-tube ion mobility spectrometry is also reported along with examples of the powerful capabilities of this platform. Lastly, nano-DESI MSI is used to address the complexity in the analysis of individual skeletal muscle fibers. This collaborative project involves the development of a robust image registration approach of immunofluorescence imaging and high-spatial resolution nano-DESI MSI to obtain accurate chemical maps specific to each fiber type. The developments described in this thesis are key to understanding the dynamic metabolic processes on a molecular level with an unprecedented specificity and sensitivity.</p> <p>  </p>

Nano-DESI Mass spectrometry imaging of eicosanoids in mouse kidney tissue using selected ion monitoring. Nano-DESI mass spectrometry imaging (MSI) is a technique for label-free spatial and molecular characterization of surfaces and biological samples. Eicosanoids are lipid mediators derived from eicosapolyenoic acid- products of arachidonic acid oxidation. Eicosanoids have been of interest to the medical field for many years. Major focus on this lipid class came from the development of nonsteroidal anti-inflammatory drugs (NSAIDs), some of these including aspirin, naproxen, ibuprofen, and acetaminophen work by blocking either the formation or the effects of eicosanoids. These lipids also play important roles in various body functions (cardiovascular, renal, gastrointestinal, neuronal) and as mediators of inflammation, asthma, fever, pain, hypertension, and stroke. Typically, eicosanoids occur in subnanomolar concentrations, despite their high level of bioactivity, which makes them significantly more difficult to analyze via direct mass spectrometry. Here, selected ion monitoring (SIM) is used to increase the signal-to-noise of the identified eicosanoids compared to a broadband full scan mode.

No description available.

This dissertation elucidates the instrumentation and application of a hybrid ambient ionization source, laser electrospray mass spectrometry (LEMS), for the direct analysis and molecular imaging of biological tissue without matrix deposition. In LEMS, laser pulses from a Ti:Sapphire laser amplifier (60 fs, 800 nm, and 1 mJ) interact with surface analytes and transfer them from the condensed phase into the gas phase without the requirement of either exogenous matrix or endogenous water in the sample. The laser vaporized analytes are captured and ionized by an electrospray source, and finally detected by a mass analyzer. It was found that a turn-key, robust femtosecond fiber laser with longer wavelength, longer duration, and lower pulse energy at 1042 nm, 425 fs, and 50 µJ, respectively, provided comparable results with the Ti:Sapphire laser. Vaporization of intact, dried or aqueous cytochrome c and lysozyme samples was demonstrated by the fiber laser. A charge states distribution at lower charge states indicating folded conformation of proteins and the hemoglobin α subunit-heme complex from whole blood was observed. Endogenous anthocyanins, sugars, and other metabolites were detected and revealed the anticipated metabolite profile for the flower petal and leaf samples by the fiber laser. Phospholipids, especially phosphatidylcholine, were identified from a fresh mouse brain section sample. These lipid features were suppressed in both the fiber laser and Ti:Sapphire LEMS measurement in the presence of optimal cutting temperature compounds which are commonly used in animal tissue cryosectioning. This dissertation also details the design of an automated mass spectrometry imaging source based on the Ti:Sapphire LEMS. The laser, translation stage, and mass analyzer are synchronized and controlled using a customized user interface to enable step-by-step scanning of the area of interest on a given tissue sample. The imaging source is coupled with a high resolution accurate mass quadrupole time-of-flight (QTOF) mass analyzer with tandem mass analysis capability. A lateral resolution of 60 µm was demonstrated on a patterned ink film by LEMS imaging. Plant metabolites including sugar and anthocyanins were directly imaged from a leaf sample. Small metabolites, lipids and proteins were simultaneously imaged from a single tissue section of a pig liver sample. Biomarkers of blood-brain barrier damage and traumatic brain injury (TBI) that occurred during the injury were detected and imaged from a TBI mouse brain. The loading values from principal component analysis (PCA) were shown to be useful for identification of features of interest from the large LEMS imaging dataset. / Chemistry

博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

Desorption Electrospray Ionization Mass spectrometry Imaging (DESI-MSI) is an area of great interest and a promising tool in the field of chemical imaging. It is a powerful, label-free technique, which can determine, map and visualize different molecular compounds on a sample surface. The amount of information acquired in a single DESI-MSI experiment is enormous compared to other techniques, as it can simultaneously detect different compounds with their spatial distribution on the surface. The experiment can be used to produce two-dimensional and three-dimensional images. Chapter 2 focuses on the design and optimization of the setup for performing DESI-MS imaging on various substrates. The proposed setup was tested for its lateral spatial resolution. To provide proof-of-concept of the design, preliminary tests were performed to generate images from commercial thin layer chromatographic plates and photographic paper. Chapter 3 focuses on demonstrating the compatibility of novel microfabricated Thin Layer Chromatography plates (M-TLC plates) for detection with DESI-MSI.

<p dir="ltr">Mass spectrometry imaging (MSI) is a powerful technique for visualizing the distribution of molecules within biological samples. Advancements in MSI instrumentation and computational tools have enabled the impactful applications of this technique across various fields including clinical research, drug discovery, forensics, microbiology, and natural products. Nanospray desorption electrospray ionization (nano-DESI), an ambient localized liquid extraction ionization technique, has proven valuable to the MSI community. Nano-DESI has been used for imaging of various molecules in biological samples including drugs, metabolites, lipids, N-linked glycans, and proteins.</p><p dir="ltr">My research has been focused on expanding the sensitivity and specificity of nano-DESI for biomolecular imaging. One of the newly developed methods employs ammonium fluoride NH₄F as a solvent additive to enhance the sensitivity of nano-DESI for the analysis of lipids in negative ionization mode. Secondly, methods were developed for the spatial mapping of isobaric and isomeric species in biological tissues by implementing nano-DESI MSI on a triple quadrupole (QqQ) mass spectrometer. This work used multiple reaction monitoring (MRM) mode of a QqQ with unit mass resolution to separate isobaric lipid species that require high mass resolving power and imaging of isomeric low-abundance species in tissue sections. Next, I demonstrate nano-DESI as a liquid extraction technique for imaging of N-linked glycans within biological tissue sections. Lastly, the spatial distribution of eicosanoids and specialized pro-resolving mediators (SPMs) in a mouse model for acetaminophen-induced liver injury (AILI) provides insights into the inflammation and resolution phases of AILI. Collectively, these developments have advanced the sensitivity, chemical specificity, and molecular coverage of nano-DESI for imaging of different classes of molecules in biological tissues.</p>

No description available.

Le cholestérol est l'une des molécules biologiques indispensable au bon fonctionnement de la plupart des organismes vivants, y compris chez l’homme. Cette molécule se trouve en abondance dans des tissus cérébraux et joue trois rôles principaux dans l'organisme. C’est un constituant (composant) essentiel de la membrane cellulaire qui sert à maintenir l’intégrité et la fluidité des cellules. Le cholestérol est aussi un élément déclencheur pour la production d’hormones stéroïdiennes comme les hormones sexuelles et la vitamine D. Finalement, il contribue à la production des acides biliaires par le foie. Dans cette étude, une méthode analytique de quantification absolu du cholestérol dans sections tissulaires de cerveau de souris par IMS a été développée. Pour ce faire, dans un premier temps des courbes d’étalonnage faites à partir de concentrations croissantes de cholesterol-d7 ont été réalisé en dopant directement des sections minces d’homogénat de cerveau. Par la suite, un étalon interne de stigmastérol (un stérol naturel d’origine exclusivement végétale) a été utilisé pour normaliser les signaux en provenance du cholestérol et du cholestérol-d7. Finalement, les analyses ont été effectué en utilisant une méthode IMS préalablement développée au laboratoire pour la détection spécifique et l’imagerie du cholestérol par désorption ionisation laser assistée par l’argent. L’étalon interne a été utilisé ici pour réduire les erreurs instrumentales, et les résultats avant et après normalisation montrent le rôle fonctionnel de cette méthode dans l’amélioration de la linéarité de la courbe d’étalonnage et, en conséquence, la mesure précise du cholestérol dans des échantillons analysés. / Cholesterol is one of the biological molecules essential for the proper functioning of most living organisms, including humans, and accurate quantification of cholesterol has many potential implications. This molecule is found abundantly in the brain and plays three main roles in the body. It is an essential component of the cell membrane which serves to maintain the integrity and fluidity of cells. Cholesterol is also a chemical trigger for the production of various steroid hormones such as sex hormones and vitamin D. Ultimately, it helps the liver to produce bile acids. A greater understanding of cholesterol and of its role in the body may directly impact our understanding of these processes. In this study, an analytical method for the absolute quantification of cholesterol in the mouse brain slices by IMS was developed. To achieve this calibration curves made from increasing concentrations of cholesterol-d7 were first performed by doping them on thin sections of brain homogenate. Subsequently, stigmasterol (a natural sterol of exclusively plant origin) was used as an internal standard to normalize the signals from cholesterol and cholesterol-d7 was evenly deposited over all analyzed sections. Finally, the analyzes were performed using an IMS method previously developed in the laboratory for the specific detection and imaging of cholesterol by silver-assisted laser ionization desorption. The internal standard was used here to reduce instrument errors, and the before and after normalization results show the functional role of this method in improving the linearity of the calibration curve and, therefore, the accurate measurement of cholesterol in the analyzed samples.