Low-dose Scanning Transmission Electron Microscopy Research Articles

Atomic-resolution STEM image denoising by total variation regularization.

Atomic-resolution electron microscopy imaging of solid-state material is a powerful method for structural analysis. Scanning transmission electron microscopy (STEM) is one of the actively used techniques to directly observe atoms in materials. However, some materials are easily damaged by the electron beam irradiation, and only noisy images are available when we decrease the electron dose to avoid beam damages. Therefore, a denoising process is necessary for precise structural analysis in low-dose STEM. In this study, we propose total variation (TV) denoising algorithm to remove quantum noise in an STEM image. We defined an entropy of STEM image that corresponds to the image contrast to determine a hyperparameter and we found that there is a hyperparameter that maximizes the entropy. We acquired atomic-resolution STEM image of CaF2 viewed along the [001] direction and executed TV denoising. The atomic columns of Ca and F are clearly visualized by the TV denoising, and atomic positions of Ca and F are determined with the error of ±1 pm and ±4 pm, respectively.

Software electron counting for low-dose scanning transmission electron microscopy

The performance of the detector is of key importance for low-dose imaging in transmission electron microscopy, and counting every single electron can be considered as the ultimate goal. In scanning transmission electron microscopy, low-dose imaging can be realized by very fast scanning, however, this also introduces artifacts and a loss of resolution in the scan direction. We have developed a software approach to correct for artifacts introduced by fast scans, making use of a scintillator and photomultiplier response that extends over several pixels. The parameters for this correction can be directly extracted from the raw image. Finally, the images can be converted into electron counts. This approach enables low-dose imaging in the scanning transmission electron microscope via high scan speeds while retaining the image quality of artifact-free slower scans.

Imaging the Surface of LiMn2O4 with Low-Dose STEM

The surface of materials is one of the most important aspects of electrochemistry. The surface is where all critical charge transfers and catalytic interactions occur. For Li-ion batteries, the surface of the electrodes dictates the reactivity with the electrolyte, the ability for Li-ions to shuttle between the bulk and the electrolyte, and the rate at which the ions can transfer, all of which has an effect on rate-capability and cyclability. LiMn2O4 (LMO) is a promising cathode material with a high-energy density and a high-rate capability, but it is plagued with a cyclability problem based on a surface effect. In the LMO system the main contributor to cycling degradation is the Mn disproportionation reaction (2Mn3+ = Mn2+ + Mn4+) which creates soluble Mn2+ that is lost to solution. We have used a combination of high-angle annular dark-field (HAADF) aberration-corrected scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS) to confirm the underlying spinel structure, and we have found, in as-processed LMO, a surface structure composed of Mn3O4 and a lithium-rich Li1+xMn2O4 subsurface layer which occurs as a result of surface reconstruction. We have also identified that oxygen loss is the mechanism by which the surface reconstruction occurs, but the question still remains, is the reconstruction of the material native or is it an effect of the electron beam? Oxide materials can be sensitive to electron-beam-induced phase changes so it is important to lower the electron dose low enough so that a determination can be made whether any impurity phases observed are an effect of the material or an effect of the electron beam. This research answers that question by imaging the surface of LMO with low-dose STEM by reducing the electron beam to pseudo-non-destructive levels. This work serves as an example of the importance of low-dose STEM as a critical technique for correctly identifying transformations and reconstructions in oxide or related materials.

Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy.

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, Escherichia coli and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD50 = 30 e(-)/nm(2), which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the E. coli membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD50. On the other hand, with a cumulative dose below LD50 (and lower SNR), it was still possible to visualize the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution.

Live Bacterial Physiology Visualized with 5 nM Resolution using Scanning Transmission Electron Microscopy

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus in minimal medium without compromising cell viability using a liquid flow cell in conjunction with low-dose Scanning Transmission Electron Microscopy (STEM). To preserve viability, cells and phage were positively stained with a low concentration of uranyl acetate in minimal phosphate medium to create contrast, and imaged with low-dose electron irradiation in a microfluidic liquid flow cell 2 μm thick. Under these conditions, it was established that the lethal dose LD50 = 30 per nm2·frame, which coincidentally also signals the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution inferred from Monte Carlo simulations, images of the Escherichia coli membrane, flagella and the bacteriophage (P1) were acquired with 5 nm resolution, but using a dose that exceeded LD50. On the other hand, the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, was visualized with 5 nm resolution with a cumulative dose below LD50.

Metastable ultrathin crystal in thermally grown SiO2 film on Si substrate

A silicon dioxide film on a silicon substrate is the most essential element in semiconductor devices and various advanced materials. We have elucidated the atomic structure of SiO2 films using low-dose scanning transmission electron microscopy (STEM). We have visualized a metastable crystalline SiO2 layer near a silicon substrate, which was not revealed in previous studies probably due to the vitrification caused by electron irradiation. Our experimental results also suggest a crystallographic nature of various surface oxides.

High-resolution low-dose scanning transmission electron microscopy

During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

Characterization of cryosectioned cell suspensions by STEM

To exploit the improved resolution and sensitivity that is now available for biological microanalysis it is crucial to optimize the specimen preparation techniques. This requires rapid freezing and ultrathin cryosectioning to a thickness of around 100 nm. Recently, it has been shown that low-dose scanning transmission electron microscopy (STEM) when applied to such specimens can yield structural detail which is comparable to that from conventional thin plastic sections. After the water is removed from the cryosections by freeze drying in vacuum, the dark-field STEM signal provides a high contrast image of the dry mass distributions of the specimen. In this paper we demonstrate the quality of ultrastructure that is achievable for suspensions of cultured cells which are ideal systems to study since the culture medium can be readily controlled. The method is illustrated by application to the protozoan parasite, Giardia lamblia.G. lamblia trophozoites, cultured in TYI-S-33 growth medium, were centrifuged at low speed to obtain a very high density suspension of cells.