Hole-free Phase Plate Research Articles

Comparative image simulations for phase-plate transmission electron microscopy

Numerous physical phase plates (PP) for phase-contrast enhancement in transmission electron microscopy (TEM) have been proposed and studied with the hole-free or Volta PP having a high impact and interest in recent years. This study is concerned with comparative TEM image simulations considering realistic descriptions of various PP approaches and samples from three different fields of application covering a large range of object sizes. The simulated images provide an illustrative characterization of the typical image appearance and common artifacts of the different PPs and the influence of simulation parameters especially important for PP simulations. A quantitative contrast analysis shows the superior phase-shifting properties of the hole-free phase plate for biological applications and the benefits of adjustable phase plates. The application of PPs in high-resolution TEM imaging, especially of weak-phase objects such as (atomically thin) 2D materials, is shown to increase image interpretability. The software with graphical user interface written and used for the presented simulations is available for free usage.

Phase plates in the transmission electron microscope: operating principles and applications.

In this paper, we review the current state of phase plate imaging in a transmission electron microscope. We focus especially on the hole-free phase plate design, also referred to as the Volta phase plate. We discuss the implementation, operating principles and applications of phase plate imaging. We provide an imaging theory that accounts for inelastic scattering in both the sample and in the hole-free phase plate.

Enhancement of low-spatial-frequency components by a new phase-contrast STEM using a probe formed with an amplitude Fresnel zone plate

Electron microscopy is a powerful tool for visualizing the shapes of sub-nanometer objects. However, Contrast Transfer Function (CTF) principally restricts lower frequency components in the image. To overcome this problem, phase-plate techniques have been proposed and currently Hole Free Phase Plate (HFPP) and Volta Phase Plate (VPP) are widely used especially for biological specimens to retrieve low frequency information of the sample’s potential distributions. In this report, we have developed a new phase-contrast scanning transmission electron microscope (STEM) in which a probe beam including side robes is formed with an amplitude Fresnel zone plate (FZP) and the interference patterns produced by the zero and first order diffracted waves generated by the FZP are detected. We name it FZP Phase Contrast STEM (FZP-PC-STEM) hereinafter. The amplitude FZP was manufactured by using focused ion beam (FIB) equipment, and the diffraction data were collected by using diffraction imaging technique. The validity of our proposed optical model was confirmed by comparing experimental and simulated images. Observations of carbon nanotube (CNT) bundles by using this method showed that the contrast of low-spatial-frequency components in the CNT image was significantly enhanced. This method does not, in principle, require the post-image processing used in the diffraction imaging method, and it can be easily introduced into pre-existing equipment without major modifications. The stability and robustness of the FZP inserted in condenser system were also confirmed during long-time operation. We expect that the FZP-PC-STEM will be widely applicable to high-contrast observations of low-Z samples with simple and easy operation.

Toward the quantitative the interpretation of hole-free phase plate images in a transmission electron microscope.

We present progress toward the quantitative interpretation of phase contrast images obtained using a hole-free phase plate (HFPP) in a transmission electron microscope (TEM). We consider a sinusoidal phase grating test object composed of ~5 nm deep groves in a ~13 nm thick amorphous silicon membrane. The periodic grating splits the beam current into direct beam and diffracted side beams in the focal plane of the imaging lens, where the HFPP is located. The physical separation between the beams allows for a detailed study of the HFPP phase shift evolution and its effect on image contrast. The residual phase shift of the electron beam footprint on the phase plate was measured by electron holography and used as input to image simulations that were compared to experimental data. Our results confirm that phase contrast is established by the phase difference between the direct and side beams, which we can estimate by fitting the image contrast evolution in time with an analytical formula describing the image intensity of a sinusoidal strong phase object. We also observed contrast reversal and frequency doubling of the grating image with time, which we interpret as the phase contrast arising from the interference between side beams becoming dominant. Another observation is the lateral displacement of the image fringes, which can be accounted for by a phase difference between the side beams.

Investigation of hole-free phase plate performance in transmission electron microscopy under different operation conditions by experiments and simulations

Hole-free phase plates (HFPPs), also known as Volta phase plates, were already demonstrated to be well suited for in-focus transmission electron microscopy imaging of organic objects. However, the underlying physical processes have not been fully understood yet. To further elucidate the imaging properties of HFPPs, phase shift measurements were carried out under different experimental conditions. Both positive and negative phase shifts occur depending on the diameter of the zero-order electron beam and the HFPP film temperature. The analysis of Thon ring patterns of an amorphous carbon test sample reveals that the phase-shifting patch can be significantly larger than the size of the zero-order beam on the HFPP film. An HFPP was used for in-focus phase contrast imaging of carbon nanotube (CNT) bundles under positive and negative phase-shifting conditions. The comparison of experimental and simulated images of CNT bundles gives detailed information on the phase shift profile, which depends on the spatial frequency in the vicinity of the zero-order beam. The shape of the phase shift profile also explains halo-like image artifacts that surround the imaged objects.

Magnetic textures in a hexaferrite thin film and their response to magnetic fields revealed by phase microscopy

We investigated magnetic textures in a Sc-doped hexaferrite film by means of phase microscopy with a hole-free phase plate in a transmission electron microscope. In a zero magnetic field, the stripe-shaped magnetic domains coexist with magnetic bubbles. The magnetization in both magnetic domains was oriented perpendicular to the film and the domain walls have an in-plane magnetization. In the remnant state at 9.2 mT, a number of magnetic bubbles were formed with the formation of stripe-shaped magnetic domains, and the out-of-plane component in the stripe-shaped domains gradually appeared as the film thickness increased. As the film thickness increased further, the magnetic bubbles with clockwise or counterclockwise spin helicities formed a triangular lattice. These results in the remnant state suggest that the domain-wall energy in the magnetic bubble domains is lower in the thicker region.

A new cryo-EM system for single particle analysis

A new cryo-EM system has been investigated for single particle analysis of protein structures. The system provides parallel illumination of a highly-coherent 300 kV electron beam from a cold-field emission gun, and boosts image contrast with an in-column energy filter and a hole-free phase plate. It includes motorized cryo-sample loading and automated liquid-nitrogen filling for cooling multiple samples. In this study, we describe gun and electron beam characteristics, and demonstrate the suitability of this system for single particle reconstructions. The performance of the system is tested on two examples, a spherical virus and apoferritin. GUI programs have also been developed to control and monitor the system for correct illumination, imaging with less ellipticity and steady magnification, and timing of flashing and liquid-nitrogen filling. These programs are especially useful for efficient application of the system to single particle cryo-EM.

Hole free phase plate tomography for materials sciences samples

We report, for the first time, the three dimensional reconstruction (3D) of a transistor from a microprocessor chip and roughness of molecular electronic junction obtained by electron tomography with Hole Free Phase Plate (HFPP) imaging. The HFPP appears to enhance contrast between inorganic materials and also increase the visibility of interfaces between different materials. We demonstrate that the degree of enhancement varies depending on material and thickness of the samples using experimental and simulation data.

Observation of FeGe skyrmions by electron phase microscopy with hole-free phase plate

We report application of hole-free phase plate (HFPP) to imaging of magnetic skyrmion lattices. Using HFPP imaging, we observed skyrmions in FeGe, and succeeded in obtaining phase contrast images that reflect the sample magnetization distribution. According to the Aharonov-Bohm effect, the electron phase is shifted by the magnetic flux due to sample magnetization. The differential processing of the intensity in a HFPP image allows us to successfully reconstruct the magnetization map of the skyrmion lattice. Furthermore, the calculated phase shift due to the magnetization of the thin film was consistent with that measured by electron holography experiment, which demonstrates that HFPP imaging can be utilized for analysis of magnetic fields and electrostatic potential distribution at the nanoscale.

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.

Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy.

Phase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the "hole-free" phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

C3-P-01Cryo-TEM Applications with Zernike and Hole-free Phase Plate

With contrast enhancement technique, cryo-transmission electron microscopy (cryo-TEM) can be more advantageous. A Zernike phase plate (ZPP) and a hole-free phase plate (HFPP) are reported to have capability to enhance contrast [1, 2, 3, 4]. The ZPP is made of thin amorphous carbon film with a center hole, and the HFPP is made of thinner carbon film with no hole. In this study, we report the results with these phase plates on cryo-microscopic techniques such as cryo-TEM tomography and single particle analysis. With ZPP and HFPP, images in a close-to-focus condition exhibited higher contrast compared to defocus phase-contrast images (see Fig. A-C). The image with HFPP shows reduced fringe contrast (Fig. C), compared to the image with ZPP (Fig. B). In addition, the HFPP does not require any alignment procedure due to lack of a center hole. Thus, the data acquisition with HFPP could be easier than that with ZPP. [1] R Danev and K Nagayama, Ultramicroscopy 88 (2001) 243-252. [2] M Malac et al, Ultramicroscopy 118 (2012), 77-89. [3] M Malac et al, U.S. Patent US 8,785,850. [4] M Malac et al, JP. Patent 2011-151019. Figure: Cryo-TEM images of ice-embedded GroEL particles acquired with a few microns of defocus (A), ZPP (B) and HFPP (C). The images with ZPP and HFPP were taken with a close-to-focus condition. C2-P-07 doi:10.1093/jmicro/dfv303

Magnetic Imaging with a Novel Hole-Free Phase Plate

One of the main interests in phase plate imaging is motivated by a decrease in irradiation dose needed to obtain desired signal to noise ratio, a result of improved contrast transfer [1]. The decrease in irradiation improves the imaging of biological materials [2]. Here we demonstrate that phase plate imaging of magnetic samples (phase objects), using a hole-free phase plate (HFPP) [3], is superior to conventional Fresnel imaging with significantly improved signal to background ratio under in-focus or near-in-focus conditions.

Magnetic imaging with a Zernike-type phase plate in a transmission electron microscope

We demonstrate the use of a hole-free phase plate (HFPP) for magnetic imaging in transmission electron microscopy by mapping the domain structure in PrDyFeB samples. The HFPP, a Zernike-like imaging method, allows for detecting magnetic signals in-focus to correlate the sample crystal structure and defects with the local magnetization topography, and to evidence stray fields protruding from the sample. Experimental and simulated results are shown and are compared with conventional Fresnel (out-of-focus) images without a phase plate. A key advantage of HFPP imaging is that the technique is free from the reference wave distortion from long-range fields affecting electron holography.

Practical hole-free phase plate imaging: principles, advantages and pitfalls

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Convenient contrast enhancement by a hole-free phase plate

Decrease of the irradiation dose needed to obtain a desired signal-to-noise ratio can be achieved by Zernike phase-plate imaging. Here we present results on a hole-free phase plate (HFPP) design that uses the incident electron beam to define the center of the plate, thereby eliminating the need for high precision alignment and with advantages in terms of ease of fabrication. The Zernike-like phase shift is provided by a charge distribution induced by the primary beam, rather than by a hole in the film. Compared to bright-field Fresnel-mode imaging, the hole-free phase plate (HFPP) results in two- to four-fold increase in contrast, leading to a corresponding decrease in the irradiation dose required to obtain a desired signal-to-noise ratio. A local potential distribution, developed due to electron beam-induced secondary-electron emission, is the most likely mechanism responsible for the contrast-transfer properties of the HFPP.