Microscopy and Analysis

Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

Piezoeletricidade e ferroeletricidade em aminoácido glicina

Doutoramento em Nanociências e NanotecnologiaBioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and switchable polarization at room temperature. Here we study piezoelectricity and ferroelectricity in the smallest amino acid glycine, representing a broad class of non-centrosymmetric amino acids. Glycine is one of the basic and important elements in biology, as it serves as a building block for proteins. Three polymorphic forms with different physical properties are possible in glycine (α, β and γ), Of special interest for various applications are non-centrosymmetric polymorphs: β-glycine and γ-glycine. The most useful β-polymorph being ferroelectric took much less attention than the other due to its instability under ambient conditions. In this work, we could grow stable microcrystals of β-glycine by the evaporation of aqueous solution on a (111)Pt/Ti/SiO2/Si substrate as a template. The effects of the solution concentration and Pt-assisted nucleation on the crystal growth and phase evolution were characterized by X-ray diffraction analysis and Raman spectroscopy. In addition, spin-coating technique was used for the fabrication of highly aligned nano-islands of β-glycine with regular orientation of the crystallographic axes relative the underlying substrate (Pt). Further we study both as-grown and tip-induced domain structures and polarization switching in the β-glycine molecular systems by Piezoresponse Force Microscopy (PFM) and compare the results with molecular modeling and computer simulations. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of applied voltage and pulse duration. The domain shape is dictated by both internal and external polarization screening mediated by defects and topographic features. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that β-glycine is a uniaxial ferroelectric with the properties controlled by the charged domain walls which in turn can be manipulated by external bias. Besides, nonlinear optical properties of β-glycine were investigated by a second harmonic generation (SHG) method. SHG method confirmed that the 2-fold symmetry is preserved in as-grown crystals, thus reflecting the expected P21 symmetry of the β-phase. Spontaneous polarization direction is found to be parallel to the monoclinic [010] axis and directed along the crystal length. These data are confirmed by computational molecular modeling. Optical measurements revealed also relatively high values of the nonlinear optical susceptibility (50% greater than in the z-cut quartz). The potential of using stable β-glycine crystals in various applications are discussed in this work.Piezo e ferroeléctricos biorgânicos são materiais que estão a atrair para si uma importância crescente por força da sua compatibilidade intrínseca com ambientes biológicos e uma biofuncionalidade aliada a um forte efeito piezoeléctrico e polarização controlada, a temperature ambiente. Aqui estudamos a piezo e ferroelectricidade no mais pequeno aminoácido, a glicina, representando uma ampla classe de aminoácidos nao-centrosimétricos. A glicina é um elemento básico e extremamente importante em biologia, uma vez que serve de unidade base de construção para proteínas. Três formas polifórmicas com diferentes propriedades são possíveis na glicina (α, β e γ). De especial interesse para várias aplicações são as estruturas não-centrosimétricas: β-glycina e γ-glycina. A mais interessante β-polimorfa está a ser alvo de uma atenção reduzida, comparativamente às outras, por motivos de uma maior instabilidade a temperatura ambiente. Neste trabalho, Podemos crescer microcristais estáveis de glicina-β pela evaporação da solução aquosa num substrato (111)Pt/Ti/SiO2/Si que funciona como "template". Os efeitos da concentração da solução e da nucleação Pt-assistida no crescimento do cristal e evolução da fase foram estudados com recurso à difracção Raio-X e espectroscopia Raman. Adicionalmente, a técnica de "spin-coating" foi utilizada para a fabricação de nano-ilhas de glicina-β altamente alinhadas, com a orientação dos eixos cristalográficos normalizada pelo substrato de Pt. Estudamos a indução de domínios estruturais por meio da ponta do AFM e a variação da polarização nos sistemas moleculares da β-glicina através da técnica PFM (Microscopia de Piezo Força), comparando os resultados obtidos com modelação molecular e simulações computacionais. Mostramos que a β-glycina é de facto um piezoeléctrico à temperatura ambiente e a polarização pode ser controlada por aplicação de uma tensão a cortes não polares. A dinâmica destes domínios complanares é estudada como função da tensão aplicada e duração do pulso. A forma do domínio é ditada pela polarização interna e externa, cujo rastreio é mediado por defeitos e características topográficas. A teoria termodinâmica é aplicada para explicar a propagação dos domínios induzidos pela ponta do AFM. As nossas descobertas sugerem que a β-glycina é um ferroeléctrico uniaxial com propriedades controladas pelas fronteiras dos domínios (electronicamente carregadas), que em seu turno podem ser manipuladas por tensão externa. Adicionalmente, propriedades ópticas não-lineares da β-glycina foram investigadas por um método de segunda geração harmonica (SHG). Este método confirmou que a simetria axial é preservada em cristais crescidos sem pós-tratamento, reflectindo a esperada simetria P21 da fase β. A direcção da polarização espontânea mostrou ser paralela ao eixo monoclínico [010] e direccionada no comprimento do cristal. Estes dados foram confirmados por modelação computacional molecular. Medições ópticas revelaram também um valor relativamente elevado para a susceptabilidade óptica não-linear (50% maior que no quartzo com corte em z). O pontencial uso de cristais de β-glycina estáveis em diversas aplicações são também discutidos

Water-Responsive, Peptide-Based Crystals

Water-responsive (WR) materials  that exert significant forces in response to changing hydration levels are receiving growing interest due to their potential applications, including use as actuators for energy harvesting devices, artificial muscles, and soft robotics. Reported examples include biological and synthetic materials with abilities to efficiently convert the chemical potential of water into mechanical actuations. However, these systems are typically complex, and consequently, their WR mechanisms are not well-understood thus preventing rational design and optimization. This thesis demonstrates the design and development of WR peptide crystals that mimic and enhance understanding of natural WR systems. The peptide crystals, with intrinsic water channels in nanoscale, have the ability to swell and shrink in response to changes in relative humidity (RH), and their WR behavior is strongly dictated by their building blocks’ chemical nature and consequent organization. The crystals also exhibit outstanding mechanical properties, structural stability, and high energy density. The modulation of peptide-sequence-dependent properties allowed us to identify key parameters that contribute to functionality during transition processes, such as dual network domains, the importance of structured water, and strengthening of hydrogen bonds, hierarchical order, order/disorder domains, and intrinsic porosity. These findings open up a magnitude of possibilities for programing simple, bioinspired peptide materials

Determination of collagen fiber orientation in histological slides using Mueller microscopy and validation by second harmonic generation imaging.

International audienceWe studied the azimuthal orientations of collagen fibers in histological slides of uterine cervical tissue by two different microscopy techniques, namely Mueller polarimetry (MP) and Second Harmonic Generation (SHG). SHG provides direct visualization of the fibers with high specificity, which orientations is then obtained by suitable image processing. MP provides images of retardation (among other polarimetric parameters) due to the optical anisotropy of the fibers, which is enhanced by Picrosirius Red staining. The fiber orientations are then assumed to be those of the retardation slow axes. The two methods, though fully different from each other, provide quite similar maps of average fiber orientations. Overall, our results confirm that MP microscopy provides reliable images of dominant fiber orientations at a much lower cost that SHG, which remains the "gold standard" for specific imaging of collagen fibers using optical microscopy

Raman spectroscopy for skin cancer diagnosis and characterisation of thin supported lipid films

Raman spectroscopy is a powerful tool in oncological imaging. Optical biopsies in which an accurate diagnosis of the tumour areas is spectroscopically performed are especially interesting for application to skin cancer treatments. In the first part of this dissertation a study on automated Raman spectral imaging allowed accurate diagnosis and delineation of the borders of a common type of skin cancer, basal cell carcinoma (BCC). Automated detection and imaging of BCC in skin sections excised during surgery was performed by combining Raman micro-spectroscopy with supervised multivariate mathematical algorithms based on linear discriminant analysis (LDA). The model allowed 90±9% sensitivity and 85±9% specificity in BCC detection. Raman spectral images based on the LDA model were created and compared with the gold-standard of the conventional histopathological diagnoses resulting in excellent agreement. Additional studies on the ability of the model in discriminating between BCC and hair follicles produced accurate diagnoses. In this thesis instrumental implementation and design of a Raman spectral imaging prototype aiming to reduce the acquisition time required to build the Raman spectral images was developed. High sensitivity variants of Raman spectroscopy such as surface enhanced Raman spectroscopy (SERS) are known to enable optical detection down to single molecules and can be applied to thin supported lipid research. The combination of SERS with a complementary topographic technique simultaneously synchronised adds to the chemical information the morphology of the sample surface. In the second part of this thesis simultaneous atomic force microscopy (AFM) and SERS characterisation of thin (≈15-20 nm) supported films of arachidic acid and cationic phospholipids on sapphire/silver substrates was successfully achieved. Supports were fabricated with nanosphere lithographic procedures and allowed enhancement of the weak Raman signals from the amphiphilic films by a maximum factor of ×10^8

Raman spectroscopy for skin cancer diagnosis and characterisation of thin supported lipid films

Raman spectroscopy is a powerful tool in oncological imaging. Optical biopsies in which an accurate diagnosis of the tumour areas is spectroscopically performed are especially interesting for application to skin cancer treatments. In the first part of this dissertation a study on automated Raman spectral imaging allowed accurate diagnosis and delineation of the borders of a common type of skin cancer, basal cell carcinoma (BCC). Automated detection and imaging of BCC in skin sections excised during surgery was performed by combining Raman micro-spectroscopy with supervised multivariate mathematical algorithms based on linear discriminant analysis (LDA). The model allowed 90±9% sensitivity and 85±9% specificity in BCC detection. Raman spectral images based on the LDA model were created and compared with the gold-standard of the conventional histopathological diagnoses resulting in excellent agreement. Additional studies on the ability of the model in discriminating between BCC and hair follicles produced accurate diagnoses. In this thesis instrumental implementation and design of a Raman spectral imaging prototype aiming to reduce the acquisition time required to build the Raman spectral images was developed. High sensitivity variants of Raman spectroscopy such as surface enhanced Raman spectroscopy (SERS) are known to enable optical detection down to single molecules and can be applied to thin supported lipid research. The combination of SERS with a complementary topographic technique simultaneously synchronised adds to the chemical information the morphology of the sample surface. In the second part of this thesis simultaneous atomic force microscopy (AFM) and SERS characterisation of thin (≈15-20 nm) supported films of arachidic acid and cationic phospholipids on sapphire/silver substrates was successfully achieved. Supports were fabricated with nanosphere lithographic procedures and allowed enhancement of the weak Raman signals from the amphiphilic films by a maximum factor of ×10^8

Assessment of the nanomechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy

Coronary atherosclerosis is a major cause of mortality and morbidity worldwide. Despite its systemic nature, atherosclerotic plaques form and develop at “predilection” sites often associated with disturbed biomechanical forces. Therefore, computational approaches that analyse the biomechanics (blood flow and tissue mechanics) of atherosclerotic plaques have come to the forefront over the last 20 years. Assignment of appropriate material properties is an integral part of the simulation process. Current approaches for derivation of material properties rely on macro-mechanical testing and are agnostic to local variations of plaque stiffness to which collagen microstructure plays an important role. In this work we used Atomic Force Microscopy to measure the stiffness of healthy and atherosclerotic coronary arteries and we hypothesised that are those are contingent on the local microstructure. Given that the optimal method for studying mechanics of arterial tissue with this method has not been comprehensively established, an indentation protocol was firstly developed and optimised for frozen tissue sections as well as a co-registration framework with the local collagen microstructure utilising the same tissue section for mechanical testing and histological staining for collagen. Overall, the mechanical properties (Young’s Modulus) of the healthy vessel wall (median = 11.0 kPa, n=1379 force curves) were found to be significantly stiffer (p=1.3410-10) than plaque tissue (median=4.3 kPa, n=1898 force curves). Within plaques, lipid-rich areas (median=2.2 kPa, n=392 force curves) were found significantly softer (p=1.4710-4) than areas rich in collagen, such as the fibrous cap (median=4.9 kPa, n=1506 force curves). No statistical difference (p=0.89) was found between measurements in the middle of the fibrous cap (median=4.8 kPa, n=868 force curves) and the cap shoulder (median=5.1 kPa, n=638 force curves). Macro-mechanical testing methods dominate the entire landscape of material testing techniques. Plaques are very heterogenous in composition and macro-mechanical methods are agnostic to microscale variations in plaque stiffness. Mechanical testing by indentation may be better suited to quantify local variations in plaque stiffness, that are potent drivers of plaque rupture.Open Acces