Nanoparticle size and 3D shape measurement by electron tomography: An Inter-Laboratory Comparison.

Electron tomography (ET) has been used for quantitative measurement of shape and size of objects in three dimensions (3D) for many years. However, systematic investigation of repeatability and reproducibility of ET has not been evaluated in detail. To assess the reproducibility and repeatability of a protocol for measuring size and three-dimensional (3D) shape parameters for nanoparticles (NPs) by ET, an inter-laboratory comparison (ILC) has been performed. The ILC included six laboratories and six instruments models from three instrument manufacturers following a standard measurement protocol. A technical specification describing the normative steps of the protocol is published by the International Standards Organization (ISO). Gold NPs with 30 nm nominal diameter contained within a rod-shaped carbon support were measured. The use of a rod-shaped sample support eliminated the missing wedge effect in the experimental tilt series of projected images for improved quantification. A total of 443 NPs were initially measured by NRC-NANO and then 115 out of the 443 NPs were measured by five other labs to compare measurands such as the Volume (V), maximum Feret diameter (Fmax), minimum Feret diameter (Fmin), volume-equivalent diameter (Deq) and aspect ratio (Frat) of the NPs. The results of the five labs were compared with the results obtained at NRC-NANO. The maximum disagreement in measurements of Fmin and Fmax obtained by the participating labs did not exceed 7 %. The measured Deq was between 27.5 nm and 30.3 nm in agreement with the NP manufacturer's specification (28 nm-32 nm). In addition to the above, the influence of the missing wedge effect and beam-induced NP movement was quantified based on the differences of the results between labs.

Fast and accurate defocus modulation for improved tunability of cryo-EM experiments.

Current data collection strategies in electron cryo-microscopy (cryo-EM) record multiframe movies with static optical settings. This limits the number of adjustable parameters that can be used to optimize the experiment. Here, a method for fast and accurate defocus (FADE) modulation during movie acquisition is proposed. It uses the objective lens aperture as an electrostatic pole that locally modifies the electron beam potential. The beam potential variation is converted to defocus change by the typically undesired chromatic aberration of the objective lens. The simplicity, electrostatic principle and low electrical impedance of the device allow fast switching speeds that will enable per-frame defocus modulation of cryo-EM movies. Researchers will be able to define custom defocus 'recipes' and tailor the experiment for optimal information extraction from the sample. The FADE method could help to convert the microscope into a more dynamic and flexible optical platform that delivers better performance in cryo-EM single-particle analysis and electron cryo-tomography.

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.

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.

Transmission electron microtomography in soft materials.

This review summarizes the recent advances in three-dimensional (3D) imaging techniques and their application to polymer nanostructures, for example, microphase-separated structures of block copolymers. We place particular emphasis on the method of transmission electron microtomography (electron tomography for short; hereafter abbreviated as ET). As a result of recent developments in ET, truly quantitative 3D images of polymer nanostructures can now be obtained with subnanometer resolution. The introduction of scanning optics in ET has made it possible to obtain large amounts of 3D data from micrometer-thick polymer specimens by using conventional electron microscopes at a relatively low accelerating voltage, 200 kV. Thus, ET covers structures over a wide range of thicknesses, from a few nanometers to several hundred nanometers, which corresponds to quite an important spatial range for hierarchical polymer nanostructures. ET provides clear 3D images and a wide range of new structural information that cannot be obtained using other methods. Information traditionally derived from conventional microscopy or scattering methods can be directly acquired from 3D volume data. ET is a versatile technique that is not restricted to only polymer applications; it can also be used as a powerful characterization tool in energy applications such as fuel cells.

Dependence of beam broadening on detection angle in scanning transmission electron microtomography.

It has been shown that scanning transmission electron microtomography (STEMT) is quite effective for observing specimens with thicknesses on the order of micrometers in three dimensions (3D). In STEMT, the specimen is scanned using a focused electron beam, and the electrons from the convergence point are detected at the detector placed at a certain detection angle. Until recently, a wide detection angle corresponding to the mode often called the dark-field (DF) mode was mainly used. Although the detection angle can vary and is one of the crucial experimental factors in STEMT, its effect on 3D reconstruction has never been discussed from either an experimental or a theoretical viewpoint. Moreover, the effectiveness of another mode of electron tomography, transmission electron microtomography (TEMT), is not clear. In the present study, a polymeric specimen, an acrylonitrile butadiene styrene resin, with a thickness of ~1 mum and a fixed volume was observed using three different modes, namely, TEMT, small detection-angle STEMT referred to as bright-field STEMT, and DF-STEMT, in order to examine their advantages and disadvantages by observing multiple scattering of electrons inside the specimen.