Alexander Orchowski

Artefacts in electron holography

Abstract In off-axis electron holography artefacts may arise specific to the method. In particular at high resolution they must be taken into account to obtain reliable results after correction of aberrations. In the following we describe some artefacts which we have found to be most essential so far.

In situ observation of electron beam-induced phase transformation of CaCO3 to CaO via ELNES at low electron beam energies.

It is demonstrated that energy-filtered transmission electron microscope enables following of in situ changes of the Ca-L2,3 edge which can originate from variations in both local symmetry and bond lengths. Low accelerating voltages of 20 and 40 kV slow down radiation damage effects and enable study of the start and finish of phase transformations. We observed electron beam-induced phase transformation of single crystalline calcite (CaCO3) to polycrystalline calcium oxide (CaO) which occurs in different stages. The coordination of Ca in calcite is close to an octahedral one streched along the <111> direction. Changes during phase transformation to an octahedral coordination of Ca in CaO go along with a bond length increase by 5 pm, where oxygen is preserved as a binding partner. Electron loss near-edge structure of the Ca-L2,3 edge show four separated peaks, which all shift toward lower energies during phase transformation at the same time the energy level splitting increases. We suggest that these changes can be mainly addressed to the change of the bond length on the order of picometers. An important pre-condition for such studies is stability of the energy drift in the range of meV over at least 1 h, which is achieved with the sub-Ångström low-voltage transmission electron microscope I prototype microscope.

X-ray Microscopy: The Cornerstone for Correlative Characterization Methods in Materials Research and Life Science

X-ray tomography has emerged as a powerful imaging technique that obtains 3D structural information from opaque samples under a variety of conditions and environments [1, 2]. It has rapidly become an accepted laboratory technique offering quantitative information in both the materials sciences and life sciences. Here we present ways in which non-destructive 3D volumetric information, obtained via laboratory nanoscale and sub-micron X-ray microscopy (XRM) are increasingly used to probe scientific questions as a complement to Electronand Light-based microscopy methods. These correlative methods, relating to XRM, provide an opportunity to study materials evolution at multiple length scales in 3D and utilize this information to inform or guide postmortem analysis to be most efficient.

Correlative large volume imaging across scales

For correlative imaging of large volumes integrated workflows allowing shuttling of samples between different imaging modalities would be ideal. X-ray microscopy (XRM) is one modality, which in principle can bridge the gap between macroscopic and microscopic world. Here we have started to assess how XRM, light microscopy (LM) and electron microscopy (EM) might be integrated to gain comprehensive information about biological samples extended in 3D. In CLEM, correlated LM and EM, fluorescence LM is commonly used to define a certain functional state, which is then put into structural context using EM. That this is also possible for XRM has been shown for single cells grown on TEM grids which were plunge frozen and analyzed by cryo-XRM [1]. For tissue samples such an approach is not possible because samples thicker than 200 micrometers cannot be vitrified. In that case a different workflow is required, starting with a chemical fixation. In LM and XRM samples may be then imaged directly in aqueous medium, XRM being able to yield voxel sizes down to 350 nm. However, if ultrastructural resolution in the range of few nanometers is required XRM needs to be complemented by EM. There are several options to achieve that for large volumes [2] based on SEM imaging such as serial blockface or focussed ion beam scanning electron microscopy (SBFSEM or FIBSEM) or array tomography (AT). SBFSEM is optimal for connectomics where whole brains need to be imaged at high resolution. In other areas of cell and developmental biology the biggest part of the sample surface being imaged may not be interesting for the question being addressed, only a minute part of it may contain the target structure. So the question of how to identify this target is of eminent importance. Here we employ XRM to identify a rare event such as the formation of an immunological synapse (IMS) or a rare structure such as the neuromuscular junction (NMJ) within a large volume. We are using the new solution ZEISS Atlas 5 (Carl Zeiss Microscopy GmbH) which offers a sample centric correlative environment fusing all available 2D and 3D data of the sample from various modalities. It contains modules for automated SEM large area imaging and targeted crossbeam (XB) nanotomography. Based on a large volume XRM dataset it thus permits an efficient approach for nanometer scale analysis of identified buried features of interest using Crossbeam technology. Figure 1 illustrates typical workflows for large volume samples such as muscle tissue or cell pellets. Important is the definition of a reference framework, which is inherent to the samples of interest. This “sample coordinate system” needs to be mapped seamlessly from one imaging technique to the next, which then allows for a fast and accurate navigation of the sample in 3D.

Advanced deflector elements for high-throughput electron optical systems

We have successfully produced and outfitted in-lens deflector elements which can be used for off-axis aberration correction in high throughput electron optics. A thorough analysis of mechanical tolerances, the study of the effect of mechanical tolerances on the imaging performance, and the comparison of calculated and measured deflection fields indicate the capability of such deflector elements for reaching the demands of high throughput electron optical devices.

Electron projection lithography: progress on the electron column modules for SCALPEL high-throughput/alpha exposure tools

We report on the realization of the electron column modules for the SCALPEL HT/Alpha EPL systems, designed to demonstrate high wafer throughput at resolutions at and below 100 nm. We describe our highly modular setup of each electron optical component targeted at maximum flexibility and enabling a fast and smooth evolution towards higher throughput and resolution. By applying strict design and process rules we were able to set-up the complete production flow from the design, construction and manufacturing of the components of the ferrite/dielectric deflector based projection optics up to established qualification schemes within less than one year. Crucial for the overall tool performance is the timely availability of system alignment and metrology strategies. Here we adapt state-of-the-art techniques form light optical lens manufacturing to a maximum amount. We discuss our metrology and alignment approach based on aerial image analysis combined with extensive electron optical imaging simulations and present first theoretical and experimental sub-100 nm results.

Progress on the realization of the electron column modules for SCALPEL high-throughput/alpha electron projection lithography tools

With the production of the first High-Throughput Alpha Tools, Electron Beam Projection Lithography (EPL) is entering the commercialization phase. Here, we report on the realization of the electron column modules for the SCALPEL HT/Alpha EPL systems, designed to demonstrate high wafer throughput at resolutions at and below 100 nm. We describe our highly modular setup of each electron optical component targeted at maximum flexibility and enabling a fast and smooth evolution towards higher throughput and resolution. By applying strict design and process rules we were able to set-up the complete production flow from the design, construction and manufacturing of the components of the ferrite/dielectric deflector based projection optics up to established qualification schemes within less than one year. A crucial point for the overall tool performance is the timely availability of system alignment and metrology strategies. Here we adapt state-of-the-art techniques from light optical lens manufacturing to a maximum amount. We discuss our metrology and alignment approach based on aerial image analysis combined with extensive electron optical imaging simulations and present first theoretical and experimental sub-100 nm results.