The work discussed in this dissertation is aimed at creating novel approaches to chemical imaging that ultimately allow for submicron resolution. This goal has been approached from two direction using laser based desorption and coupling it with an AFM using apertureless tip-enhanced laser ablation/ionization. The second direction was through the development a new approach to thermal desorption based mass spectrometry experiments by using a proximal probe to spatially desorb the surface and ionizing the plume of neutrals using a secondary ionization source at atmospheric pressure. The thermal desorption approach allows for the easy scaling of the technique all the way from the millimeter to the nanometer regime. In the nanometer regime an AFM platform with silicon based heating AFM probes is used to locally desorb material from nanometer sized craters. The final work in this thesis focused on trying to improve laser based desorption through a secondary ionization of the neutrals plume by capturing the laser desorption plume into a liquid and then electrospaying the solution into a MS. The added benefit of being able to capture the desorption plume into a liquid is the ability to carry out post sampling processing of the captured analyte via high performance liquid chromatography. The ability to clean up a sample via HPLC also allow for the detection of isobaric compounds as well as trace level materials which otherwise would be obscured by matrix effects in complicated sample matrixes like tissues. This application of laser desorption with a secondary ionization by capture into a liquid could be envisioned to be applied to AFM based laser desorption techniques where boosting the ionization efficiency is crucial for signal detection.

Master of Science / Food Science Institute / David L. Wetzel / Grain processing and handling requires quality determinations to ensure wholesome products that meet or surpass legal standards and specifications required by the end consumer. Near infrared spectroscopy has proven to be a useful and versatile tool to enable grain processers to make adjustments as needed. Near infrared chemical imaging also provides spatial information within the image and relative composition of chemically distinct components within the product. The potential use of chemical imaging to determine extraneous material in bread baking quality flour was addressed. A specimen preparation technique was developed. Insect fragment spiked specimens were imaged to determine their imaging effectiveness for application near the allowable limit of insect fragment concentration. Imaging was achieved using indium antimonide array detection of diffusely reflected radiation. The detector array of 81,920 pixels collected radiation from an area of 30.72 mm by 38.4 mm with a pixel size of 120 µm². Spectra were collected simultaneously from each pixel without moving parts by scanning with a liquid crystal tunable filter. Partial least squares analysis of each pixel within the sample allowed a summation of the insect quantity. The chemical structural distinction of chitin in the high protein matrix of the insect residue was in contrast to the non-digested carbohydrate residue in the lesser protein matrix of the flour. The method developed provided a linear response for a concentration range from approximately half the allowable limit to twice the limit for two insects that commonly contaminate flour. For the two insects studied the slopes are comparable with a slight off-set over a practical working range, so that insect concentration can be determined independently of species recognition.

Orientador: Ronei Jesus Poppi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T17:44:22Z (GMT). No. of bitstreams: 1 Sabin_GuilhermePost_D.pdf: 9789223 bytes, checksum: 0f451c3cf56a345b71358f196448c6d9 (MD5) Previous issue date: 2013 / Resumo: A indústria farmacêutica está em constante busca por novas tecnologias analíticas capazes de fornecer informações relevantes sobre produtos e processos industriais. Esta tese sugere novas possibilidades no estudo de medicamentos através de imagens químicas obtidas por microespectroscopia na região do infravermelho próximo e as seguintes ferramentas quimiométricas: MLR, CLS, MCR e PLS. Os trabalhos estão divididos em quatro tópicos e contribuem para o avanço analítico neste campo de conhecimento. Na primeira aplicação, foi realizado um estudo da similaridade entre imagens obtidas por técnicas quimiométricas distintas. O trabalho mostra como estimar limites de confiabilidade para a concentração por pixel de imagem. Assim, foi possível diminuir o tempo de aquisição de imagens mantendo a confiabilidade analítica no estudo de comprimidos de carbamazepina. O segundo estudo mostra uma aplicação onde a vantagem de segunda ordem foi requerida. Este estudo traz uma abordagem nova sobre o reconhecimento de padrões de imagem na caracterização de produtos farmacêuticos. Na terceira aplicação, o objetivo foi acompanhar o desenvolvimento industrial de um produto farmacêutico com baixo teor de ativo, auxiliando no processo de decisão em termos de micro-homogeneidade da formulação. O estudo mostrou que a concentração local pode ser explorada como vantagem analítica. Na quarta aplicação, foi abordado pela primeira vez na literatura, o estudo da estrutura de pellets farmacêuticos através de informações químicas obtidas por espectroscopia de imagem química na região do infravermelho próximo. Este trabalho abre alternativas para o estudo de medicamentos de entrega controlada. / Abstract: The pharmaceutical industry is constantly searching for new analytical technologies capable of providing relevant information about products and industrial processes. This thesis suggests new possibilities in the study of drugs by using chemical images obtained by microspectroscopy in the near infrared region and the following chemometric tools: MLR, CLS, MCR e PLS. The work is divided into four topics and it contributes to the advancement in the field of analytical knowledge. In the first application, a study was performed about the similarity between images obtained by different chemometric techniques. The work demonstrates how to estimate concentration reliability limits per image pixel. Thus, it was possible to shorten the time of image acquisition while maintaining analytical reliability in the study of carbamazepine tablets. The second study presents an application where the advantage of the second order was required. This study presents a new approach to the recognition of image patterns in the characterization of pharmaceuticals. In the third application, the aim was to follow the industrial development of a pharmaceutical product with a low active content, assisting in the decision about the process in terms of micro-homogeneity of the formulation. The study showed that the local concentration can be exploited for analytical advantage. In the fourth application, addressed in a publication for the first time, is the study of the structure of pharmaceutical pellets through chemical information obtained by near infrared chemical imaging. This work opens up alternatives for the study of controlled delivery drugs. / Doutorado / Quimica Analitica / Doutor em Ciências

La thèse propose une méthodologie d’analyse rapide basée sur l’étude de l’image hyperspectrale Raman d’un produit pharmaceutique falsifié sous forme solide afin, d’une part d’identifier les substances présentes, et d’autre part estimer la teneur du principe actif dans l’échantillon sans étalonnage préalable.La présence de produits pharmaceutiques falsifiés est un véritable enjeu de santé publique. Ce type de produits de santé est facilement disponible sur internet, et beaucoup d’exemples montrent leur dangerosité. De nombreuses techniques sont disponibles pour analyser ces produits et ainsi participer à la lutte contre la falsification de médicament. La combinaison de ces techniques analytiques permet une caractérisation approfondie de l’échantillon. Cependant, peu de techniques analytiques procurent l’ensemble des informations chimiques.L’imagerie chimique Raman est une technique qui répond aux exigences requises pour l’analyse de produits falsifiés sous forme solide. En effet, cette technique peu destructive permet de réutiliser l’échantillon pour des analyses complémentaires. L’imagerie chimique Raman combine les trois disciplines de spectroscopie Raman, microscopie et chimiométrie. Cette technique réalise des mesures successives de spectres Raman sur des zones adjacentes couvrant la surface de l’échantillon. Elle intègre donc des informations spatiales et spectrales. Les méthodes chimiométriques dites de résolution (MCR-ALS et DCLS) analysent le jeu de spectres pour extraire des informations qualitatives (détection des spectres purs du mélange) et des informations quantitatives (estimation de la concentration de la substance active). La méthodologie a été optimisée et validée avec des échantillons préparés en laboratoire, puis appliquée à des échantillons réels authentiques et falsifiés. La sensibilité de la méthode qualitative a été démontrée par la détection d’un principe actif antibiotique à la teneur de 0,3% m/m dans un comprimé à visée anabolisante. De plus la méthode a été capable de détecter les substances utilisées pour le traitement de dysfonctions sexuelles (sildénafil, tadalafil, vardénafil, dapoxétine). Malgré une forte émission de fluorescence, la méthode a réussi à discriminer les 3 sels de clopidogrel (hydrogénosulfate, bésilate et chlorhydrate). L’analyse quantitative directe sur des échantillons de Viagra® et de Plavix® a été jugée convenable avec une déviation de la teneur entre -15% et +24%. Cette déviation est considérée acceptable pour évaluer le risque sanitaire pour le patient et alerter les autorités de santé.Dans le cadre de l’analyse des produits falsifiés, il a été démontré que la micro-spectroscopie Raman associée aux méthodes chimiométriques permet de réaliser un « screening spectroscopique » des composants de l’échantillon, d’identifier les substances chimiques, de visualiser leur distribution sur la surface de l’échantillon et d’estimer leur teneur par « quantification directe ». / The thesis proposes a rapid methodology of analysis based on the Raman hyperspectral image study of a solid form falsified pharmaceutical product in order to identify the substances in the sample and to estimate the content of the active ingredient in the sample without prior calibration.The presence of falsified pharmaceuticals is a real public health issue. This type of products is easily available on the internet, and many examples show their dangerousness. Many techniques are available for the analysis of these products and thus participate in the fight against drug falsification. The combination of these analytical techniques allows a comprehensive characterization of the sample. However few analytical techniques provide all the chemical information. Raman chemical imaging is a technique that meets the requirements for the analysis of falsified products in solid form. Indeed, this non-destructive technique makes it possible the reuse of the sample for additional testing. Raman chemical imaging combines the three disciplines of Raman spectroscopy, microscopy and chemometrics. This technique performs successive measurements of Raman spectra on adjacent location covering the surface of the sample. It therefore collects spatial and spectral information. The so-called resolution chemometric methods analyse the set of spectra in order to extract qualitative information (detection of pure spectra in the mixture) and quantitative information (estimate of the concentration of the chemical substance). The methodology was optimized and validated with samples prepared in the laboratory, and then applied to genuine and falsified real samples. The sensitivity of the qualitative method was demonstrated with the detection of an antibiotic active ingredient at a content of 0.3% m/m in an anabolic tablet. Moreover, the method was able to distinguish substances used for the treatment of sexual dysfunctions (sildenafil, tadalafil, vardenafil, dapoxetine). Despite a high fluorescence emission, the method successfully discriminated the 3 salts of clopidogrel (hydrogen sulfate, besylate and hydrochloride). Direct quantitative analysis of samples of Viagra® and Plavix® was found to be appropriate with a deviation between -15% and +24%. This deviation is considered acceptable to assess the health risk to the patient and to alert health authorities.For the analysis of falsified products, it has been demonstrated that Raman micro-spectroscopy combined with chemometric methods allows to perform a "spectroscopic screening" of the components in the sample, to identify chemical substances, to visualize their distribution on the sample surface and to estimate their content by "direct quantification".

No description available.

Volumetric optical microscopy has the advantages of quantitative and global measurement of three-dimensional (3D) biological specimens with high spatial resolution and minimum invasion. However, current volumetric imaging technologies based on light transmission, scattering or fluorescence cannot reveal specimen’s chemical distribution that brings insights to study the chemical events in organisms and their metabolism, functionality, and development. Stimulated Raman scattering (SRS) microscopy allowing visualization of chemical contents based on their intrinsic molecular vibrations is an emerging imaging technology to provide rapid label-free volumetric chemical imaging. This dissertation describes three methodologies for developing advanced volumetric SRS imaging technologies to address the challenges of imaging in vivo samples, imaging speed, and axial resolution. In the first methodology, SRS volumetric imaging is enabled by axially scanning the laser foci for sectioning different depth layers. In Chapter 2, we utilize a piezo objective positioner to drive the objective. Combining with the tissue clearance technique, we realize volumetric SRS imaging up to 500 µm depth in brain tissues showing the potential for 3D staining-free histology. The limitations of piezo scanning are slow speed and disturbance to in vivo samples while rapidly scanning the objective. To tackle the limitations, in Chapter 3, we develop a remote-focusing volumetric SRS microscope based on a deformable mirror and adaptive optics optimization, allowing focal scanning without physically moving the objective or sample. We demonstrate in vivo monitoring of chemical penetration in human sweat pores. In the second methodology, instead of axially scanning the laser foci, the SRS volumetric imaging is enabled by projection imaging with extended depth-of-focus (DOF) beams such as Bessel beams and low numerical-aperture beams. The extended DOF beams integrate SRS signals along the propagation direction to form projection images; thus, a single lateral scan obtains the volumetric chemical information, significantly increasing the volumetric imaging speed for measuring chemical content over a large volume. In Chapter 4, we describe a stimulated Raman projection microscope for fast quantitation of chemicals in a 3D volume. However, projection imaging intrinsically loses axial resolution. We addressed the limitation by developing SRS projection tomography. Mimicking computed tomography, the axial information is reconstructed by angle-dependent projection images obtained by sequentially rotating the sample in a capillary glass tube within the SRS focus. Nevertheless, sample rotation is complicated and not compatible with in vivo samples. To address the difficulty, in Chapter 5, we develop tilted-angle-illuminated stimulated Raman projection tomography which utilizes tilted-angle beams with a tilted angle respected to the optical axis of the objective to obtain angle-dependent projections. This scheme is free of sample rotation and enables fast projection scanning for pushing the imaging speed. The calibration approach and vector-field back-projection algorithm are developed for the multi-view tomographic reconstruction. In the third methodology, we improve the spatial resolution in miniature volumetric SRS imaging via the innovation of metasurface photonics. In developing an SRS endoscope for volumetric chemical imaging inside the human body, the axial resolution deteriorates due to chromatic and monochromatic aberrations induced by poorly made miniature objective lenses. In Chapter 6, we develop a silicon metasurface tailored for compensating the phase errors between the pump and Stokes wavelengths of a singlet refractive lens. Integrating the metasurface with the refractive lens, the hybrid achromatic metalens is compact and provides nearly diffraction-limit resolution, demonstrating a way for developing high resolution chemical imaging endoscopy.

Master of Science / Department of Grain Science and Industry / D.L. Wetzel / Chemical imaging enables displaying the distribution of different substances within a field of view based on their fundamental vibrational frequencies. Mid-IR bands are generally strong and feature direct correlation to chemical structure, while near IR spectra consist of overtones and combinations of mid-IR bands. Recently, mid-IR microspectroscopy has enabled determination of the relative substitution of hydroxyl groups with the modifying agent for individual waxy maize starch granules by using synchrotron source. The brightness and non-divergence of the synchrotron source and confocal masking enabled obtaining individual spectra with 5 [mu]m[superscript]2 masking and 1 [mu]m raster scanned steps. Each 1 [mu]m step results from the coaddition of hundreds of scans and lengthy data collection is required to produce data. The recent breakthrough at the Synchrotron Research Center uses a multi-beam synchrotron source combined with a focal plane array microspectrometer. This major improvement in localized detection of the modifying agent within single waxy maize starch granules is the increased efficiency of focal plane array detection and an effective spatial resolution of 0.54 [mu]m. Mixtures of granular solids represent an analytical challenge due to the range of heterogeneity and homogeneity within samples. Near IR imaging provides deeper sample penetration allowing for solid mixture analysis. However, the broad, overlapping bands present in the near IR necessitates statistical data treatment. This requires imaging specimens representative of the individual components to create spectral libraries for classification of each component. Partial least squares analysis then allows characterization and subsequent pixel analysis provides quantitative results. The primary break system for wheat milling was studied as it is key in releasing endosperm to be further ground into fine flour in subsequent processes. The mass balance of endosperm throughout individual unit processes was determined by obtaining flow rates of incoming and outgoing millstreams and calculating endosperm content through pixel identification. The feed milling industry requires the use of a tracer to determine adequate mixing and mix uniformity to limit the time and energy in processing. Near IR imaging allows individual components of a formula feed to serve as a self-tracer, eliminating the need of an inorganic tracer.

Isolierte Molekülcluster können durch Fourier-Transformations-Infrarotspektroskopie von Überschallexpansionen untersucht werden. Es wird gezeigt, wie Multischlitzdüsen verschiedener Geometrien sowohl zur Aufklärung des Strukturverhaltens von Wasseraggregaten bei thermischer Anregung als auch zur Erzeugung von Mischclustern bei gleichzeitiger Unterdrückung von Isotopenaustauschreaktionen eingesetzt werden können. Zusätzlich wurde ein Aufbau zur chemischen Bildgebung von Expansionen entwickelt und zur ortsaufgelösten Visualisierung von Aggregationsprozessen sowie Dichte- und Temperaturentwicklungen verwendet.

L'histologie tridimensionnelle (3D) est un nouvel outil avancé de cancérologie. L'ensemble du profil chimique et des caractéristiques physiologiques d'un tissu est essentiel pour comprendre la logique du développement d'une pathologie. Cependant, il n'existe aucune technique analytique, in vivo ou histologique, capable de découvrir de telles caractéristiques anormales et de fournir une distribution3D à une résolution microscopique. Nous présentons ici une méthode unique de microscopie infrarouge (IR) à haut débit combinant une correction d'image automatisée et une analyse ultérieure des données spectrales pour la reconstruction d'image 3D-IR. Nous avons effectué l'analyse spectrale d'un organe complet pour un petit modèle animal, un cerveau de souris avec une tumeur de gliome implantée. L'image 3D-IR est reconstruite à partir de 370 coupes de tissus consécutives et corrigée à l'aide du tomogramme à rayons X de l'organe pour une analyse quantitative précise du contenu chimique. Une matrice 3D de spectres IR 89 x 106 est générée, ce qui nous permet de séparer la masse tumorale des tissus cérébraux sains en fonction de divers paramètres anatomiques,chimiques et métaboliques. Nous démontrons pour la première fois que des paramètres métaboliques quantitatifs (glucose, glycogène et lactate) peuvent être extraits et reconstruits en 3D à partir des spectres IR pour la caractérisation du métabolisme cérébral / tumoral (évaluation de l'effet de Warburg dans les tumeurs). Notre méthode peut être davantage exploitée en recherchant l'ensemble du profil spectral, en distinguant différents points de repère anatomiques dans le cerveau.Nous le démontrons par la reconstruction du corps calleux et de la région des noyaux gris centraux du cerveau. / Three-dimensional (3D) histology is a new advanced tool for cancerology. The whole chemical profile and physiological characteristics of a tissue is essential to understand the rationale of pathology development. However, there is no analytical technique, in vivo or histological, that is able to discover such abnormal features and provide a 3D distribution at microscopic resolution.Here, we introduce a unique high- throughput infrared (IR) microscopy method that combines automated image correction and subsequent spectral data analysis for 3D-IR image reconstruction. I performed spectral analysis of a complete organ for a small animal model, a mouse brain with animplanted glioma tumor. The 3D-IR image is reconstructed from 370 consecutive tissue sectionsand corrected using the X-ray tomogram of the organ for an accurate quantitative analysis of thechemical content. A 3D matrix of 89 x 106 IR spectra is generated, allowing us to separate the tumor mass from healthy brain tissues based on various anatomical, chemical, and metabolic parameters. I demonstrate for the first time that quantitative metabolic parameters (glucose, glycogen and lactate) can be extracted and reconstructed in 3D from the IR spectra for the characterization of the brain vs. tumor metabolism (assessing the Warburg effect in tumors). Our method can be further exploited by searching for the whole spectral profile, discriminating different anatomical landmarks in the brain. I demonstrate this by the reconstruction of the corpus callosum and basal ganglia region of the brain.

Master of Science / Department of Grain Science and Industry / David L. Wetzel / During germination, biochemical changes occur in the wheat kernel by stimulation of enzymes and hormones, and the seed reserves are mobilized. Infrared microspectroscopy and imaging enables a localized chemical inventory, upon germination, to study the process. Frozen sections of germinated wheat mounted onto BaF[subscript]2 were mapped to produce functional group images for comparison with corresponding sections of ungerminated kernels. Relative functional group populations in the scutellum and embryonic axis were assessed before and after germination. An average 23% reduction in lipid to protein ratio was observed in the scutellum based on the comparison of 53,733 spectra. As a result of the early germination process, lipid in the scutellum was depleted to provide energy for the growing embryo. Germination of the kernels while in the field before harvest due to high humidity is known as preharvest sprouting. Preharvest sprouting has detrimental effects on the end use quality of the wheat (sprout damage) and cause economic loses. Tolerance to preharvest sprouting is highly desirable. To assist breeding program, a nondestructive near-IR chemical imaging method has been developed to test new lines for resistance to preharvest sprouting. The higher sensitivity of subsurface chemical imaging, compared with visual detection, alpha amylase determination, or viscosity testing, permits germination detection at early stages. A near-IR chemical imaging system with an InGaAs focal plane array (FPA) detector in the 1100 nm-1700 nm range was used. Kernels from six different cultivars, including HRW and HWW wheat, were exposed to moist conditions for 6, 12, 24, 36, and 48 hours. Images of each 90 kernel group were examined; kernels exposed to moisture for 36 hours were compared with kernels treated for 3 hours as a control. Each kernel was classified as sprouted or not sprouted with the criteria of log 1/R intensity at select wavelengths or select factors of principle component analysis (PCA) treatment of reflectance intensity data. Imaging wavelength range was expanded beyond 1700 nm to 2400 nm with the use of InSb FPA. Study for the potential for unsupervised determination in nondestructive near-IR imaging with detection wavelengths 1200-2400 is ongoing. Some preliminary results presented are encouraging.