Simon Hettler

In-Focus Electrostatic Zach Phase Plate Imaging for Transmission Electron Microscopy with Tunable Phase Contrast of Frozen Hydrated Biological Samples

Transmission electron microscopy (TEM) images of beam sensitive weak-phase objects such as biological cryo samples usually show a very low signal-to-noise ratio. These samples have almost no amplitude contrast and instead structural information is mainly encoded in the phase contrast. To increase the sample contrast in the image, especially for low spatial frequencies, the use of phase plates for close to focus phase contrast enhancement in TEM has long been discussed. Electrostatic phase plates are favorable in particular, as their tunable potential will allow an optimal phase shift adjustment and higher resolution than film phase plates as they avoid additional scattering events in matter. Here we show the first realization of close to focus phase contrast images of actin filament cryo samples acquired using an electrostatic Zach phase plate. Both positive and negative phase contrast is shown, which is obtained by applying appropriate potentials to the phase plate. The dependence of phase contrast improvement on sample orientation with respect to the phase plate is demonstrated and single-sideband artifacts are discussed. Additionally, possibilities to reduce contamination and charging effects of the phase plate are shown.

Low affinity binding of plasma proteins to lipid-coated quantum dots as observed by in situ fluorescence correlation spectroscopy

Protein binding to lipid-coated nanoparticles has been pursued quantitatively by using fluorescence correlation spectroscopy. The binding of three important plasma proteins to lipid-enwrapped quantum dots (QDs) shows very low affinity, with an apparent dissociation coefficient in the range of several hundred micromolar. Thus, the tendency to adsorb is orders of magnitude weaker than for QDs coated with dihydrolipoic acid.

Improving Fabrication and Application of Zach Phase Plates for Phase-Contrast Transmission Electron Microscopy

Zach phase plates (PPs) are promising devices to enhance phase contrast in transmission electron microscopy. The Zach PP shifts the phase of the zero-order beam by a strongly localized inhomogeneous electrostatic potential in the back focal plane of the objective lens. We present substantial improvements of the Zach PP, which overcome previous limitations. The implementation of a microstructured heating device significantly reduces contamination and charging of the PP structure and extends its lifetime. An improved production process allows fabricating PPs with reduced dimensions resulting in lower cut-on frequencies as revealed by simulations of the electrostatic potential. Phase contrast with inversion of PbSe nanoparticles is demonstrated in a standard transmission electron microscope with LaB6 cathode by applying different voltages.

Carbon contamination in scanning transmission electron microscopy and its impact on phase-plate applications

We analyze electron-beam induced carbon contamination in a transmission electron microscope. The study is performed on thin films potentially suitable as phase plates for phase-contrast transmission electron microscopy. Electron energy-loss spectroscopy and phase-plate imaging is utilized to analyze the contamination. The deposited contamination layer is identified as a graphitic carbon layer which is not prone to electrostatic charging whereas a non-conductive underlying substrate charges. Several methods that inhibit contamination are evaluated and the impact of carbon contamination on phase-plate imaging is discussed. The findings are in general interesting for scanning transmission electron microscopy applications.

Thin-Film Phase Plates for Transmission Electron Microscopy Fabricated from Metallic Glasses

Thin-film phase plates (PPs) have become an interesting tool to enhance the contrast of weak-phase objects in transmission electron microscopy (TEM). The thin film usually consists of amorphous carbon, which suffers from quick degeneration under the intense electron-beam illumination. Recent investigations have focused on the search for alternative materials with an improved material stability. This work presents thin-film PPs fabricated from metallic glass alloys, which are characterized by a high electrical conductivity and an amorphous structure. Thin films of the zirconium-based alloy Zr65.0Al7.5Cu27.5 (ZAC) were fabricated and their phase-shifting properties were evaluated. The ZAC film was investigated by different TEM techniques, which reveal beneficial properties compared with amorphous carbon PPs. Particularly favorable is the small probability for inelastic plasmon scattering, which results from the combined effect of a moderate inelastic mean free path and a reduced film thickness due to a high mean inner potential. Small probability plasmon scattering improves contrast transfer at high spatial frequencies, which makes the ZAC alloy a promising material for PP fabrication.

Charging of carbon thin films in scanning and phase-plate transmission electron microscopy

A systematic study on charging of carbon thin films under intense electron-beam irradiation was performed in a transmission electron microscope to identify the underlying physics for the functionality of hole-free phase plates. Thin amorphous carbon films fabricated by different deposition techniques and single-layer graphene were studied. Clean thin films at moderate temperatures show small negative charging while thin films kept at an elevated temperature are stable and not prone to beam-generated charging. The charging is attributed to electron-stimulated desorption (ESD) of chemisorbed water molecules from the thin-film surfaces and an accompanying change of work function. The ESD interpretation is supported by experimental results obtained by electron-energy loss spectroscopy, hole-free phase plate imaging, secondary electron detection and x-ray photoelectron spectroscopy as well as simulations of the electrostatic potential distribution. The described ESD-based model explains previous experimental findings and is of general interest to any phase-related technique in a transmission electron microscope.

Computer simulations analysis for determining the polarity of charge generated by high energy electron irradiation of a thin film

Detailed simulations are necessary to correctly interpret the charge polarity of electron beam irradiated thin film patch. Relying on systematic simulations we provide guidelines and movies to interpret experimentally the polarity of the charged area, to be understood as the sign of the electrostatic potential developed under the beam with reference to a ground electrode. We discuss the two methods most frequently used to assess charge polarity: Fresnel imaging of the irradiated area and Thon rings analysis. We also briefly discuss parameter optimization for hole free phase plate (HFPP) imaging. Our results are particularly relevant to understanding contrast of hole-free phase plate imaging and Berriman effect.

On the role of inelastic scattering in phase-plate transmission electron microscopy

The phase contrast of Au nanoparticles on amorphous-carbon films with different thicknesses is analyzed using an electrostatic Zach phase plate in a Zeiss 912 Ω transmission electron microscope with in-column energy filter. Specifically, unfiltered and plasmon-filtered phase-plate transmission electron microscopy (PP TEM) images are compared to gain insight in the role of coherence after inelastic scattering processes. A considerable phase-contrast contribution resulting from a combined elastic-inelastic scattering process is found in plasmon-filtered PP TEM images. The contrast reduction compared to unfiltered images mainly originates from zero-order beam broadening caused by the inelastic scattering process. The effect of the sequence of the elastic and inelastic scattering processes is studied by varying the position of the nanoparticles, which can be either located on top or at the bottom of the amorphous-carbon film with respect to the incident electron beam direction.

High-resolution transmission electron microscopy with an electrostatic Zach phase plate

A new method to control lattice-fringe contrast in high-resolution transmission electron microscopy (HRTEM) images by the implementation of a physical phase plate (PP) is proposed. PPs are commonly used in analogy to Zernike PPs in light microscopy to enhance the phase contrast of weak-phase objects with nm-sized features, which often occur in life science applications. Such objects otherwise require strong defocusing, which leads to a degradation of the instrumental resolution and impedes intuitive image interpretation. The successful application of an electrostatic Zach PP in HRTEM is demonstrated by the investigation of single crystalline Si and Ge samples. The influence of the Zach PP on the image formation process is assessed by analyzing the amplitudes of (111) reflections in power spectra which show a cosine-type dependence on the induced phase shift under certain conditions as predicted by theory.

A nanocrystalline Hilbert phase-plate for phase-contrast transmission electron microscopy

Thin-film-based phase-plates are applied to enhance the contrast of weak-phase objects in transmission electron microscopy. In this work, metal-film-based phase-plates are considered to reduce contamination and electrostatic charging, which up to now limit the application of phase-plates fabricated from amorphous C-films. Their crystalline structure requires a model for the simulation of the effect of crystallinity on the phase-plate properties and the image formation process. The model established in this work is verified by experimental results obtained by the application of a textured nanocrystalline Au-film-based Hilbert phase-plate. Based on the model, it is shown that monocrystalline and textured nanocrystalline phase-plate microstructures of appropriate thickness and crystalline orientation can be a promising approach for phase-contrast transmission electron microscopy.