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

A NEW METHOD OF WAVELENGTH SCANNING INTERFEROMETRY FOR INSPECTING SURFACES WITH MULTI-SIDE HIGH-SLOPED FACETS

With the development of modern advanced manufacturing technologies, the requirements for ultra-precision structured surfaces are increasing rapidly for both high value-added products and scientific research. Examples of the components encompassing the structures include brightness enhancement film (BEF), optical gratings and so forth. Besides, specially designed structured surfaces, namely metamaterials can lead to specified desirable coherence, angular or spatial characteristics that the natural materials do not possess. This promising field attracts a large amount of funding and investments. However, owing to a lack of effective means of inspecting the structured surfaces, the manufacturing process is heavily reliant on the experience of fabrication operators adopting an expensive trial-and-error approach, resulting in high scrap rates up to 50-70% of the manufactured items. Therefore, overcoming this challenge becomes increasingly valuable. The thesis proposes a novel methodology to tackle this challenge by setting up an apparatus encompassing multiple measurement probes to attain the dataset for each facet of the structured surface and then blending the acquired datasets together, based on the relative location of the probes, which is achieved via the system calibration. The method relies on wavelength scanning interferometry (WSI), which can achieve areal measurement with axial resolutions approaching the nanometre without the requirement for the mechanical scanning of either the sample or optics, unlike comparable techniques such as coherence scanning interferometry (CSI). This lack of mechanical scanning opens up the possibility of using a multi-probe optics system to provide simultaneous measurement with multi adjacent fields of view. The thesis presents a proof-of-principle demonstration of a dual-probe wavelength scanning interferometry (DPWSI) system capable of measuring near-right-angle V-groove structures in a single measurement acquisition. The optical system comprises dual probes, with orthogonal measurement planes. For a given probe, a range of V-groove angles is measurable, limited by the acceptance angle of the objective lenses employed. This range can be expanded further by designing equivalent probe heads with varying angular separation. More complicated structured surfaces can be inspected by increasing the number of probes. The fringe analysis algorithms for WSI are discussed in detail, some improvements are proposed, and experimental validation is conducted. The scheme for calibrating the DPSWI system and obtaining the relative location between the probes to achieve the whole topography is implemented and presented in full. The appraisal of the DPWSI system is also carried out using a multi-step diamond-turned specimen and a sawtooth brightness enhancement film (BEF). The results showed that the proposed method could achieve the inspection of the near-right-angle V-groove structures with submicrometre scale vertical resolution and micrometre level lateral resolution

Three-dimensional single particle tracking in a light sheet microscope

Technical development in microscopy, and particularly in fluorescence microscopy, has facilitated the investigation of ever smaller details in biological specimen. The combination of specific labeling of molecular compounds, sophisticated optical setups and sensitive detectors enables observation of single molecules. Using fast video microscopy, it is now possible to directly observe the cell’s molecular machinery at work by tracking single molecules with high spatial and temporal resolution. Single molecule tracking can reveal detailed information about the dynamics of biological processes. However, technical requirements for single molecule detection limit the depth of field to less than 1 μm. Thus, single molecule tracking is typically limited to studying phenomena in planar membranes or, in extended specimen, often relies on two dimensional projections of short trajectory fragments. The work presented here strives to overcome these limitations by combining real-time three-dimensional localization of single particles with an active feedback loop to keep a particle of interest within the observation volume. To this end, a light sheet microscopy setup was designed and assembled around a commercial microscope body. It was equipped with a fast piezo stage for axial sample positioning. Three-dimensional spatial information was encoded in the shape of the point spread function by astigmatic detection and retrieved by real-time image analysis code developed for this purpose. A novel localization metric based on cross-correlation template matching was devised to enable tracking based on a low number of photons detected per particle. During post-processing, relative axial localizations determined from the image data were combined with the piezo stage position to obtain full three-dimensional particle trajectories. Mechanical and optical properties of the setup were thoroughly characterized using appropriate test samples. A temporal resolution down to 1,12 ms was achieved. The localization precision of the method was experimentally determined by repeated imaging of immobilized fluorescent beads. The capability to track single emitters was validated in a biochemical model system. Lipids labeled with a synthetic dye molecule were incorporated in the bilayer membrane of giant unilamellar vesicles and tracked on their spherical surface. Trajectories of more than 20 s duration could be obtained at as little as 130 photons detected per frame. An analysis of the photophysical properties revealed that observation times per particle were limited not by failure of the tracking algorithm but by photobleaching. Applicability of the method in biological specimen was proved by tracking fluorescent nanoparticles micro-injected into C. tentans salivary gland cell nuclei for more than 270 s in several thousand frames. Subsequently, the method was applied to track mRNA and rRNA particles in C. tentans salivary gland cell nuclei. Biomolecules were specifically labeled by complementary oligonucleotides carrying up to three synthetic dye molecules. It was possible to routinely acquire trajectories of particles with a diffusion coefficient of D = 1-2 μm2/s spanning ≥ 4 s and 4-5 μm in axial direction. The longest trajectories lasted more than 16 s and covered 10 μm axially. Both, observation time and axial range, were increased by more than one order of magnitude as compared to standard 2D tracking experiments. It was thus possible to investigate mobility states not on the basis of an ensemble of short observations but for individual particles

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand