Post-acquisition image based compensation for thickness variation in microscopy section series

Serial section Microscopy is an established method for volumetric anatomy reconstruction. Section series imaged with Electron Microscopy are currently vital for the reconstruction of the synaptic connectivity of entire animal brains such as that of Drosophila melanogaster. The process of removing ultrathin layers from a solid block containing the specimen, however, is a fragile procedure and has limited precision with respect to section thickness. We have developed a method to estimate the relative z-position of each individual section as a function of signal change across the section series. First experiments show promising results on both serial section Transmission Electron Microscopy (ssTEM) data and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) series. We made our solution available as Open Source plugins for the TrakEM2 software and the ImageJ distribution Fiji

Reference-free evaluation of thin films mass thickness and composition through energy dispersive x-ray spectroscopy

In this paper we report the development of a new method for the evaluation of thin films mass thickness and composition based on the Energy Dispersive X-Ray Spectroscopy (EDS). The method exploits the theoretical calculation of the in-depth characteristic X-ray generation distribution function, $\phi$/($\rho$ z), in multilayer samples, obtained by the numerical solution of the electron transport equation, to achieve reliable measurements without the need of a reference sample and multiple voltages acquisitions. The electron transport model is derived from the Boltzmann transport equation and it exploits the most updated and reliable physical parameters in order to obtain an accurate description of the phenomenon. The method for the calculation of film mass thickness and composition is validated with benchmarks from standard techniques. In addition, a model uncertainty and sensitivity analysis is carried out and it indicates that the mass thickness accuracy is in the order of 10 $\mu$g/cm$^2$, which is comparable to the nuclear standard techniques resolution. We show the technique peculiarities in one example measurement: two-dimensional mass thickness and composition profiles are obtained for a ultra-low density, high roughness, nanostructured film.Comment: This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ENSURE grant agreement No. 647554

Estimation of sample spacing in stochastic processes

Motivated by applications in electron microscopy, we study the situation where a stationary and isotropic random field is observed on two parallel planes with unknown distance. We propose an estimator for this distance. Under the tractable, yet flexible class of Lévy-based random field models, we derive an approximate variance of the estimator. The estimator and the approximate variance perform well in two simulation studies

Automation of section acquisition for Array Tomography

Array Tomography hat großes Potential, um die dreidimensionale Struktur von Proben bis zu Nanometer Größenordnungen aufzulösen. Dabei wird eine Probe mechanisch geschnitten um so innen liegende Strukturen freizulegen. Die Schnitte schwimmen zunächst auf einer Wasseroberfläche und werden dann auf starren Substraten zur Bildaufnahme abgelegt. Die Flexibilität und Vielseitigkeit der zur Verfügung stehenden bildgebenden Verfahren ist einzigartig für Array Tomography. Zur Zeit wird eine intensive Nutzung jedoch durch den hohen Arbeitsaufwand und Anspruch an die Bedienung eingeschränkt. Existierende maschinelle Systeme zur Schnittaufnahme schränken entweder die zur Verfügung stehenden Bildgebungsverfahren oder das Probenvolumen ein. In dieser Dissertation wird ein maschinelles Verfahren zur Schnittaufnahme vorgestellt, welches die gleiche Flexibilität und Vielfältigkeit ermöglicht wie die konventionelle manuelle Schnittaufnahme. Fluidkanäle bilden ein mikrofluidisches System mit geringer Reynolds Nummer, in dem sich Schnitte und Substrat gemeinsam bewegen. Die Fluidkanäle formen sich auf der Substratoberfläche durch eine lokale Modifikation der Benetzbarkeit. Die Oberflächenfunktionalisierung wird durch Abscheiden einer hydrophoben Beschichtung und anschließender Plasmastrukturierung erreicht. Das neu entwickelte System umfasst eine maschinelle Probenausrichtung, Schnittaufnahme und Schnittüberwachung. Die Schnitte können auf den für Array Tomography üblichen Substraten abgelegt und somit mit einer Vielzahl von mikroskopischen Verfahren untersucht werden. Durch die maschinelle Schnittaufnahme können große Volumen effizient geschnitten werden, wodurch die Anwedung der Array Tomography in neuen Forschungsgebieten möglich wird. Die maschinelle Schnittaufnahme ist an zwei repräsentativen Proben mit jeweils 1000 Schnitten validiert

Characterisation Protocol for Liquid- Phase-Synthesised Graphene

Graphene, a two-dimensional honeycomb sp2 carbon lattice has received enormous attention because of the potential for various applications such as the electrodes of photovoltaic devices and batteries, next-generation flexible electronics and even antibacterial coatings. Interest in the application of graphene is mainly due to its unique physical and chemical properties, flexibility, and tuneability of the properties in graphene-based materials. However, while promising applications of graphene are being discussed, the term ‘graphene’ is often misused, and the difficulties in large-scale production of true two-dimensional graphene have further limited its applications. Methods such as top-down solution-processed exfoliation were developed to overcome the obstacles for large-scale graphene production, but these approaches do not yet produce completely delaminated and homogeneous graphene. To monitor and optimise the graphene production process, the development of a fast, standardised and reliable characterisation protocol for large-scale solution-processed graphene is therefore desirable. Among the many characteristics of graphene flakes, the nano-structural features including the lateral dimension, crystal imperfections and the thicknesses of graphene are the most important factors that affect the various properties of graphene. However, though many of the analytical techniques have continuously been improved, methods to obtain and quantify these graphene nano-structural features are still limited. This is owing to the difficulties of visualising the ultra-thin nano-flakes and the fact that many of the properties of graphene are still unknown to be used to identify the material. In this study, a characterisation protocol was proposed to quantify the fundamental nano- structural features of graphene. In all cases, the nano-structural feature was initially characterised by using the most precise technique based on direct imaging from transmission electron microscopy (TEM), the results were being used as benchmarks for the other fast but less direct methods that based on photon-probe techniques. To integrate and assess different characterisation techniques, quantification and statistical analysis of results have been used. By utilising the method proposed, it was found that the lateral dimension distribution of graphene can be rapidly obtained by Dynamic Light Scattering (DLS), especially for flakes smaller than 1000 nm. The crystalline imperfections within graphene can be obtained and quantified by conventional Raman spectroscopy, in which a simple method based on linear correlation and random sampling was proposed to indicate the source of disorder in graphene samples. The result was compared to the TEM study, and the differences were assigned to the uneven distribution of the defects in graphene flakes. The thickness of graphene was characterised via various techniques. Several empirical equations were derived in order to can be rapidly obtained the thickness of graphene. However, it may not be feasible at this stage to develop a method to accurately determine graphene thickness for large-scale characterisation. It was found that the level of graphitic character could be obtained utilising the variation of Raman 2D (G’) band, which is rather more important, and can be used to improve the graphene synthesis process. In summary, the proposed graphene characterisation protocol offers a practical method to integrate and evaluate different characterisation techniques. Also, the protocol development method can be used as a reference point, which can be applied to other materials for developing material-specific characterisation protocols. Nevertheless, it has been shown that such a graphene characterisation protocol has the ability to quantify and differentiate between inhomogeneous solution-processed graphene samples and can be used for optimising the graphene synthesis processes