3D Imaging Photocatalytically Degraded Micro- and Nanoplastics.

Microplastics and nanoplastics have been an emerging global concern, with hazardous effects on plant, animal, and human health. Their small size makes it easier for them to spread to various ecosystems and enter the food chain; they are already widely found in aqueous environments and within aquatic life, and have even been found within humans. Much research has gone into understanding micro-/nanoplastic sources and environmental fate, but less work has been done to understand their degradation. Photocatalytic degradation is a promising green technique that uses visible or ultraviolet light in combination with photocatalyst to degrade plastic particles. While complete degradation, reducing plastics to small molecules, is often the goal, partial degradation is more common. We examined microscale polyethylene (125-150 µm in diameter) and nanoscale polystyrene (~300 nm in diameter) spheres both before and after degradation using multiple imaging techniques, especially scanning electron microscopy and electron tomography. Electron tomography is able to image the 3D exterior and interior of the nanoplastics, enabling us to observe within aggregates and inside degraded spheres, where we found potentially open interior structures after degradation. These structures may result from differences in degradation and aggregation behaviour between the different plastic types, with our work finding that polyethylene microplastics typically cracked into sharp fragments, while polystyrene nanoplastics often fragmented into smoother, more curved shapes. These and other differences, along with interior and 3D surface images, provide new details on how the structure and aggregation of polyethylene microplastics and polystyrene nanoplastics changes when degraded, which could influence how the resulting worn particles are collected or treated further.

Chromosome inner structure investigation by electron tomography and electron diffraction in a transmission electron microscope.

Our understanding of the inner structure of metaphase chromosomes remains inconclusive despite intensive studies using multiple imaging techniques. Transmission electron microscopy has been extensively used to visualize chromosome ultrastructure. This review summarizes recent results obtained using two transmission electron microscopy-based techniques: electron tomography and electron diffraction. Electron tomography allows advanced three-dimensional imaging of chromosomes, while electron diffraction detects the presence of periodic structures within chromosomes. The combination of these two techniques provides results contributing to the understanding of local structural organization of chromatin fibers within chromosomes.

High-Energy Electron Scattering in Thick Samples Evaluated by Bright-Field Transmission Electron Microscopy, Energy-Filtering Transmission Electron Microscopy, and Electron Tomography.

Energy-filtering transmission electron microscopy (TEM) and bright-field TEM can be used to extract local sample thickness $t$ and to generate two-dimensional sample thickness maps. Electron tomography can be used to accurately verify the local $t$. The relations of log-ratio of zero-loss filtered energy-filtering TEM beam intensity ($I_{{\rm ZLP}}$) and unfiltered beam intensity ($I_{\rm u}$) versus sample thickness $t$ were measured for five values of collection angle in a microscope equipped with an energy filter. Furthermore, log-ratio of the incident (primary) beam intensity ($I_{\rm p}$) and the transmitted beam $I_{{\rm tr}}$ versus $t$ in bright-field TEM was measured utilizing a camera before the energy filter. The measurements were performed on a multilayer sample containing eight materials and thickness $t$ up to 800 nm. Local thickness $t$ was verified by electron tomography. The following results are reported:• The maximum thickness $t_{{\rm max}}$ yielding a linear relation of log-ratio, $\ln ( {I_{\rm u}}/{I_{{\rm ZLP}}})$ and $\ln ( {I_{\rm p}}/{I_{{\rm tr}}} )$, versus $t$.• Inelastic mean free path ($\lambda _{{\rm in}}$) for five values of collection angle.• Total mean free path ($\lambda _{{\rm total}}$) of electrons excluded by an angle-limiting aperture.• $\lambda _{{\rm in}}$ and $\lambda _{{\rm total}}$ are evaluated for the eight materials with atomic number from $\approx$10 to 79.The results can be utilized as a guide for upper limit of $t$ evaluation in energy-filtering TEM and bright-field TEM and for optimizing electron tomography experiments.

Higher-Order Structure of Human Chromosomes Observed by Electron Diffraction and Electron Tomography.

It is well known that two DNA molecules are wrapped around histone octamers and folded together to form a single chromosome. However, the nucleosome fiber folding within a chromosome remains an enigma, and the higher-order structure of chromosomes also is not understood. In this study, we employed electron diffraction which provides a noninvasive analysis to characterize the internal structure of chromosomes. The results revealed the presence of structures with 100­200 nm periodic features directionally perpendicular to the chromosome axis in unlabeled isolated human chromosomes. We also visualized the 100­200 nm periodic features perpendicular to the chromosome axis in an isolated chromosome whose DNA molecules were specifically labeled with OsO4 using electron tomography in 300 keV and 1 MeV transmission electron microscopes.

Sample thickness affects contrast and measured shape in TEM images and in electron tomograms.

We investigated the effect of nanoparticle (NP) image broadening and its contrast change dependence on a support matrix thickness in a transmission electron microscope (TEM). We measured the effect of NP size and atomic number on its image broadening. Based on the experimental TEM images we generated tomograms of NPs on four types of support matrix. The measured shape aspect ratio of the NPs in such tomograms depends on the geometry of the support matrix. For example, the aspect ratio of 6 nm NP placed on a thin film with window-frame support is 1.14, while the aspect ratio of 6 nm NP on a rod-shaped support with 910 nm diameter is 1.67 in a tomogram.

Origin of spurious intensity in vacuum near sample edge in bright field TEM images.

Bright-field transmission electron microscope (BFTEM) images exhibit spurious image intensity in the vacuum near the sample edge. The spurious intensity gradually decreases with increasing distance from the sample edge. By taking into account angular and energy loss distribution of the scattered electrons and lens aberrations, the origin of the spurious intensity of BFTEM images can be explained. The spurious intensity extent and magnitude can be significantly reduced by using either electron energy filtering or a small collection semiangle.

Higher-order structure of barley chromosomes observed by electron tomography.

The higher order structure of the metaphase chromosome has been an enigma for over a century and several different models have been presented based on results obtained by a variety of techniques. Some disagreements in the results between methods have possibly arisen from artifacts caused during sample preparation such as staining and dehydration. Therefore, we treated barley chromosomes with ionic liquid to minimize the effects of dehydration. We also observed chromosomes on a film with holes to keep pristine chromosome structure from being flattened as seen when placed on a continuous support film. A chromosome placed over a hole in a thin carbon film was mounted on a tomography holder, and its structure was observed in three dimensions (3D) using electron tomography. We found that there are periodic structures with 300-400 nm pitch along the axis in barley chromosomes. The pitch sizes are larger than those observed in human chromosomes.

NanoMi: An open source electron microscope hardware and software platform.

We outline a public license (open source) electron microscopy platform, referred to as NanoMi. NanoMi offers a modular, flexible electron microscope platform that can be utilized for a variety of applications, such as microscopy education and development of proof-of-principle experiments, and can be used to complement an existing experimental apparatus. All components are ultra-high vacuum compatible and the electron optics elements are independent from the vacuum envelope. The individual optical components are mounted on a 127 mm (5-inch) diameter half-pipe, allowing customizing of electron optics for a variety of purposes. The target capabilities include SEM, TEM, scanning TEM (STEM), and electron diffraction (ED) at up to 50 keV incident electron energy. The intended image resolution in SEM, TEM and STEM modes is ≈ 10 nm. We describe the existing components and the interfaces among components that ensure their compatibility and interchangeability. The paper provides a resource for those who consider building or utilizing their own NanoMi.

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.

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.

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.

3D observation of chromosome scaffold structure using a 360° electron tomography sample holder.

The chromosome scaffold is considered to be a key structure of the mitotic chromosome. It plays a vital role in chromosome condensation, shaping the X-shaped structure of the mitotic chromosome, and also provides flexibility for chromosome movement during cell division. However, it remains to be elucidated how the chromosome scaffold organizes the mitotic chromosome and how it supports shaping the structure of the chromosome during metaphase. Here we present a new technique that enables the observation of the chromosome scaffold structure in metaphase chromosomes from any direction, by transferring an isolated chromosome to a 360° rotational holder for electron tomography (ET). The chromosome was stained with immunogold-labeled condensin complex, one of the major chromosome scaffold proteins and then observed in three dimensions using ET. Using the locations of gold nanoparticles to visualize the underlying structure, the tomograms we obtained reveal the patterns of chromosome scaffold organization, which appears to consist of a helical structure that serves to organize chromatin loops into the metaphase chromosome.

Parameters affecting the accuracy of nanoparticle shape and size measurement in 3D.

While electron tomography can be used to visualize objects at nanoscale, it is difficult to perform reproducible quantitative measurements. Here we measure the shape and size of nanoparticles (NPs) in three dimensions (3D) using electron tomography. We evaluated the accuracy of maximum Feret diameter (Feretmax), minimum Feret diameter (Feretmini) and volume of NPs measurements from reconstructed 3D images which were obtained from data acquired with varied electron dose. We perform both simulations and experiment to clarify what factors effect on the accuracy of the NP shape measurement. Based on the results, suitable reconstruction methods and threshold for binarization were evaluated. We also report comparison results obtained on exactly the same samples in two different laboratories.

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.

Evaluation of electron tomography reconstruction methods for interface roughness measurement.

We evaluate the suitability of simultaneous iterative reconstruction technique (SIRT), filtered back projection, and simultaneous algebraic reconstruction technique methods for buried interface roughness measurements. We also investigate the effect of total electron dose distributed over the entire tilt series on measured roughness values. We investigate the applicability of the dose fractionation theorem by evaluating the effect of an increasing number of images, i.e., decreasing tilt increment size at fixed total electron irradiation dose on the quantitative measurement of buried interface roughness. The results indicate that SIRT is the most suitable method for reconstruction and a 3° to 5° angle is optimal for the roughness measurement.

Wavelet transform-based electron tomography measurement of buried interface roughness.

Interface roughness is a critical parameter determining the performance of semiconductor devices. We show that a continuous wavelet transform is useful to describe not only the magnitude of the interface roughness, but also the spatial frequencies that describe the interface. We propose a simple presentation of the results that makes it convenient to compare between interfaces. In particular, an average and maximum value wavelet profile that is obtained from a series of one dimensional wavelet transforms provides a traceable and quick survey of the results. We demonstrate the wavelet transform method using both computer simulations and by applying it to experimental data obtained by electron tomography of a test sample and to a molecular layer interface. Wavelet descriptions of the interface roughness suffers less from the presence of shot noise in the experimental data than the traditional root mean square error description of interface roughness. An increase in lateral dimensions of an interface that has large features increases the content of low spatial frequencies in wavelet transforms. In comparison, the value of root mean square error increases in an untraceable manner with the same increase in lateral dimensions on the same interface. Morse wavelets with γ = 9 and ß = 3 appear to be a suitable choice for applications in interface roughness measurement.

Thermal expansion coefficient measurement from electron diffraction of amorphous films in a TEM.

We measured the linear thermal expansion coefficients of amorphous 5-30 nm thick SiN and 17 nm thick Formvar/Carbon (F/C) films using electron diffraction in a transmission electron microscope. Positive thermal expansion coefficient (TEC) was observed in SiN but negative coefficients in the F/C films. In case of amorphous carbon (aC) films, we could not measure TEC because the diffraction radii required several hours to stabilize at a fixed temperature.

Development of computer-assisted minimal-dose system with beam blanker for TEM.

A computer-assisted minimal-dose system has been developed for the high-resolution observation of radiation-sensitive samples using a transmission electron microscope (TEM). This system consists of a CCD camera, a beam blanker and a control computer (PC) that also controls the TEM. A sample is illuminated by an electron beam only when the CCD camera takes images; otherwise, the beam blanker cuts off the electron beam. Emulated images, which are calculated from the images taken and the variable parameters of the TEM, such as magnification and sample stage position, are displayed on the control PC display. After a few times of repetition of exposures and emulations, the sample is positioned to final observation area. Subsequently to select the observation area, the system automatically adjusts the focus position from two different illuminating angle images and the TEM conditions are appropriate for taking a final image. The total electron dose before the final image is taken can be markedly decreased because the sample is irradiated by an electron beam only when images are taken. For the final image, a three-dimensional Fourier filtering method (3DFFM), which corrects the spherical aberration for the through-focus image series and enables the selection of the optimum focus condition later, is also included in our system.