Analyzing contrast in cryo-transmission electron microscopy: Comparison of electrostatic Zach phase plates and hole-free phase plates.

Phase plates (PPs) are beneficial devices to improve the phase contrast of life-science objects in cryo-transmission electron microscopy (TEM). The development of the hole-free (HF) PP, which consists of a thin carbon film, has led to impressive results due to its ease in fabrication, implementation and application. However, the phase shift of the HFPP can be controlled only indirectly. The electrostatic Zach PP uses a strongly localized and adjustable electrostatic potential to generate well-defined and variable phase shifts between scattered and unscattered electrons. However, artifacts in phase-contrast TEM images are induced by the presence of the PP rod in the diffraction plane. We present a detailed analysis and comparison of the contrast-enhancing capabilities of both PP types and their emerging artifacts. For this purpose, cryo-TEM images of a standard T4-bacteriophage test sample were acquired with both PP types. Simulated images reproduce the experimental images well and substantially contribute to the understanding of contrast formation. An electrostatic Zach PP was used in this work to acquire cryo-electron tomograms with enhanced contrast, which are of similar quality as tomograms obtained by HFPP TEM.

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.

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.

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.

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.

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.

New electrostatic phase plate for phase-contrast transmission electron microscopy and its application for wave-function reconstruction.

A promising novel type of electrostatic phase plate for transmission electron microscopy (TEM) is presented. The phase plate consists of a single microcoaxial cable-like rod with its electrode exposed to the undiffracted electrons. The emerging field is used to shift the phase of the undiffracted electrons with respect to diffracted electrons. The design overcomes the drawback of the spatial frequency-blocking ring electrode of the Boersch phase plate. First, experimental phase-contrast images are presented for PbSe and Pt nanoparticles with clearly varying phase contrast, which depends on the applied voltage and resulting phase shift of the unscattered electrons. With the new phase-plate design, we show for the first time the reconstruction of an object wave function based on a series of only three experimental phase-contrast TEM images obtained with an electrostatic phase plate.