Quantitative Automated Object Wave Restoration in High-Resolution Electron Microscopy

The main problem addressed by this dissertation is the accurate and automated determination of electron microscope imaging conditions. This enables the restoration of the object wave, which confers direct structural information about the specimen, from sets of differently aberrated images. An important member in the imaging chain is the image recording device, in many cases now a charge-coupled device (CCD) camera. Previous characterisations of these cameras often relied on the unjustified assumption that the Modulation Transfer Function (MTF) also correctly describes the spatial frequency dependent attenuation of the electron shot noise. A new theory is therefore presented that distinguishes between signal and noise transfer. This facilitates the evaluation of both properties using a detailed Monte-Carlo simulation model for the electron and photon scattering in the scintillator of the camera. Furthermore, methods for the accurate experimental determination of the signal and noise transfer functions are presented. In agreement with the Monte-Carlo simulations, experimental results for commercially available CCD cameras show that the signal transfer is significantly poorer than the noise transfer. The centrepiece of this dissertation is the development of new methods for determining the relative aberrations in a set of images and the absolute symmetric aberrations in the restored wave. Both are based on the analysis of the phase information in the Fourier domain and give each Fourier component a weight independent of its strength. This makes the method suitable even for largely crystalline samples with little amorphous contamination, where conventional methods, such as automated diffractogram fitting, usually fail. The method is then extended to also cover the antisymmetric aberrations, using combined beam tilt and focal series. The applicability of the new method is demonstrated with object wave restorations from tilt and focal series of complex inorganic block oxides and of carbon nanotubes filled with one-dimensional inorganic crystals. The latter specimens allowed for the first time a direct comparison between the phase shift in the restored object wave of a specimen with precisely known thickness and the value predicted by simulations

Analysis of Strain Relaxation, Ion Beam Damage and Instrument Imperfections for Quantitative STEM Characterizations

It is illustrated that the preparation of thin specimens from bulk materials can have significant influence on the interpretability of (S)TEM data. The results of the presented measurements show that and the elastic strain relaxation in low dimensional structures alters the overall strain state of the material – and hence affects strain measurements – as well as the contrast of STEM measurements and is generally needed to be incorporated in comparative simulation studies that involve strained structures. Furthermore, the ion beam thinning process itself can introduce – even with relatively low energies – a serious alteration of the surface which can affect the contrast of STEM measurements. Hence, the correlation to thickness measurements is complicated due to the distinct difference in scattering behaviour between (partially) amorphized surface layers in comparison with crystalline material. Although parts of these effects cannot be avoided the inclusion of amorphous pseudo-oxide layers in simulations has been shown to provide reasonable agreement with the experimental data. Furthermore, the impact of a finite electron source with limited coherence has been investigated. It can be shown that a reproduction of experimental contrast by simulation can only be achieved by the inclusion of an additional focus spread as well as a lateral point spread due to partial spatial coherence. Finally, the previous results are combined to reconstruct the three-dimensional shape of several antiphase domains within gallium phosphide grown on silicon-(001). At first the concept was demonstrated for a simple but highly strained interface and second for large structures with thousands of atomic columns. It is shown that although the contrast mechanism for annular dark-field imaging is in principle straight forward and mathematically simple, the details of atomic resolution microscopy are still very challenging. Realistic assumptions about the specimen properties and the electron optics have been shown to be of great relevance for data evaluation. It is clear that the research should be extended to the regime of low angular dark-field imaging where strain and inelastic scattering play a even more relevant role. Furthermore, it is of great importance to investigate the aforementioned practical aspects of damage layers and optical imperfections for other advanced imaging techniques like diffraction imaging. In addition, it is worth investigating in how far through focus depth section can be utilized to increase the reliability of structure restoration along the transmission direction. It is expected that the improvement of accuracy and robustness of atomic counting techniques will greatly increase the power of a (S)TEM by providing simultaneously lateral and depth information about arrangement and composition. Furthermore, it is clear that the role of high performance simulations will have an even more important role in the future

Near Field Electron Ptychography

Phase imaging in the Transmission Electron Microscope (TEM) has a long history, from the implementation of off-axis holography in TEM to Differential Phase Contrast (DPC) on the Scanning Transmission Electron Microscopy (STEM). The advent of modern computing has enabled the development of iterative algorithms which attempt to recover a phase image of a specimen from measurements of the way it diffracts an incident electron beam. One of the most successful of these iterative methods is focused probe ptychography, which relies on far field diffraction pattern measurements recorded as the incident beam is scanned through a grid of locations across the specimen. Focused probe ptychography implemented in the STEM has provided the highest resolution images available to date, allows for lens-less setups avoiding the aberrations typical in older STEMs and allows for simultaneous reconstruction of the illumination and specimen. Ptychography is computationally flexible (highly constrained), allowing for additional unknowns other than the phase of the specimen to be recovered, for example positions can be refined during reconstruction. Near field ptychography is a recent variation on ptychography that replaces the far-field diffraction data with diffraction patterns recorded in the near field, or Fresnel, region. It promises to obtain a much larger field of view with fewer diffraction patterns than focused probe ptychography. The main contribution of this thesis is the implementation of a new form of near field ptychography on the Transmission Electron Microscope (TEM), using an etched silicon nitride window to structure the electron beam. Proof-of-concept results show the method quantitatively recovers megapixel phase images from as few as 9 recorded diffraction patterns, compared to many hundreds of diffraction patterns required for focused probe ptychography. Additional sets of results show how near-field ptychography can recover extremely large fields of view, deal effectively with inelastic scattering, and accommodate several sources of uncertainty in the experimental process. Further contributions in the thesis include: experiments and results from visible-light versions of near field ptychography, which explain its limitations and practical application; a description and code for analysis tools that are used to assess phase imaging performance; DigitalMicrograph (DM) code and a data collection workflow to realise TEM-based near-field ptychography; details of the design, realisation and performance of the etched silicon nitride windows; and simulation studies aimed at furthering understanding of the frequency response of the technique. Future work is outlined, focusing on potential applications in a wide range of real-world specimens and improved TEM setups to implement near field ptychography

Photonic Technology for Precision Metrology

Photonics has had a decisive influence on recent scientific and technological achievements. It includes aspects of photon generation and photon–matter interaction. Although it finds many applications in the whole optical range of the wavelengths, most solutions operate in the visible and infrared range. Since the invention of the laser, a source of highly coherent optical radiation, optical measurements have become the perfect tool for highly precise and accurate measurements. Such measurements have the additional advantages of requiring no contact and a fast rate suitable for in-process metrology. However, their extreme precision is ultimately limited by, e.g., the noise of both lasers and photodetectors. The Special Issue of the Applied Science is devoted to the cutting-edge uses of optical sources, detectors, and optoelectronics systems in numerous fields of science and technology (e.g., industry, environment, healthcare, telecommunication, security, and space). The aim is to provide detail on state-of-the-art photonic technology for precision metrology and identify future developmental directions. This issue focuses on metrology principles and measurement instrumentation in optical technology to solve challenging engineering problems

Single-molecule stochastic localization fluorescence nanoscopy

Hasta hace unos 20 años se creía que la difracción de la luz imponía un límitefundamental de unos 200 nm a la resolución espacial de un microscopio óptico. Lasnanoscopías de fluorescencia, también llamadas microscopías de superresolución,han quebrado esta barrera y permiten en teoría alcanzar la máxima resoluciónespacial con sentido físico, es decir el tamaño mismo de la fuente de luz. En lapráctica, sin embargo, la resolución se ve limitada a unos 20 nm por efecto devariables experimentales como la relación señal-ruido y el fotoblanqueo de losmarcadores fluorescentes. Además, las nanoscopías de fluorescencia mantienenlas ventajas de la microscopía de fluorescencia tradicional, como el acceso pocoinvasivo y la alta sensibilidad y especificidad. Se denomina "localización de una molécula individual" al proceso numéricopor el cual se extrae la posición de un emisor único a partir de la medición desu patrón de intensidad. La nanoscopía por localización estocástica de moléculasindividuales (o simplemente "nanoscopía por localización") consiste en la adquisición secuencial de imágenes en las que en cada una un subconjunto estocástico delos fluoróforos de una muestra son resueltos individualmente. Como los patronesde emisión no se superponen, la precisión de localización solo depende del númerode fotones detectados de cada emisión. La imagen final se construye con las posicionesde cada fluoróforo previamente localizado. Para obtener una imagen desuperresolución es necesario que los marcadores estén separados por distanciasmenores a su imagen limitada por difracción y que éstos emitan de manera intermitente (que se enciendan y apaguen), de manera que en algún momento puedanobservarse individualmente. La nanoscopía por localización comprende a un conjunto de técnicas diferenciadas entre sí de acuerdo al mecanismo que permite elencendido y apagado de los marcadores fluorescentes. En la presente Tesis se estudian aspectos fundamentales e instrumentales dela nanoscopía por localización y se la aplica al estudio de preguntas biológicas. En el Capítulo 1 se desarrollan los conceptos fundamentales y limitaciones dela microscopía de uorescencia, y se introducen las técnicas de superresolución. En el Capítulo 2 se detallan los métodos y estado del arte de la nanoscopía porlocalización. En el Capítulo 3 se caracteriza el nanoscopio por localización conposibilidad de obtener imágenes en 3D y a dos colores de emisión, que fuera construidocomo parte del trabajo de Tesis. Finalmente, en los Capítulos 4 y 5 sedescriben aplicaciones de dicho nanoscopio en dos proyectos de relevancia biológica:el estudio en neuronas hipocampales de la estructura periódica de espectrina yla distribución espacial de proteínas presentes en la membrana del Trypanosomacruzi que median la interacción entre el parásito y el huésped.Until twenty years ago it was considered that the diffraction of light imposeda fundamental limit of around 200 nm to the resolution of an optical microscope. Fluorescence nanoscopy, also known as super-resolution uorescence microscopy,has overcome this limit. It achieves the theoretical maximum spatial resolution,which is the size of the light source itself. In practice, however, the resolution islimited to around 20 nm by experimental factors like the signal-to-noise ratio andthe photobleaching of uorescent markers. Besides, fluorescence nanoscopy maintainsthe advantages of traditional fluorescence microscopy, like its low invasivityand its high sensitivity and specificity. Single-molecule localization is the numerical process that extracts the single moleculeposition from measuring its emission pattern. Sigle-molecule stochasticlocalization nanoscopy (or simply "localization nanoscopy") consists of the sequentialacquisition of images of well separated fluorophores from a stochasticsubset of all fluorophores in the sample. As their emission patterns do not overlap,the position of each molecule can be determined with a precision only limitedby the number of detected photons. A final super-resolved image is reconstructedfrom the localizations of all the fluorophores of the sequence of images. In orderto obtain a super-resolved image, it is essential to have fluorescent markerscloser to each other than the diffraction limit. Also, they must intermittentlyswitch on and off, so that at a given point in time they can be imaged individually. Stochastic localization nanoscopy denotes a group of techniques each with adifferent mechanism for the on-off switching of the fluorophores. In this thesis we study fundamental and instrumental aspects of localizationnanoscopy and we apply it to the study of biological questions. In Chapter 1the fundamental concepts, potential and limitations of fluorescence microscopyare presented, followed by an introduction to super-resolution techniques. Thefundamental principles and methods of single-molecule localization fluorescencenanoscopy are explained in detail in Chapter 2. Chapter 3 gives a complete descriptionof the nanoscope built as part of this thesis work at the Centro de Investigaciones en Bionanociencias (CIBION), along with guidelines for its operationand an illustration of its performance. In Chapters 4 and 5 two biologicalapplications of the nanoscope are presented, that address nanoscale organizationproteins: firstly, the quantification of the periodic spectrin structure present inhippocampal neurons and secondly, the spatial distribution of proteins on theouter membrane of the Trypanosoma cruzi that mediate the parasite-host interaction. Finally, the conclusions of this thesis and future perspectives are unfoldedin Chapter 6.Fil: Barabas, Federico Martín. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina