Electron Transparent Windows Research Articles

Probing Thermoresponsive Polymerization-Induced Self-Assembly with Variable-Temperature Liquid-Cell Transmission Electron Microscopy

Summary We directly initiate and visualize the formation of polymeric nanomaterials via variable-temperature liquid-cell transmission electron microscopy (VT-LCTEM). With temperature control in the liquid cell, reversible addition-fragmentation chain transfer polymerization was thermally initiated, leading to the formation of amphiphilic block copolymers that assemble upon dispersion polymerization-induced self-assembly (PISA). In combination with traditional ex situ analyses, VT-LCTEM enabled not only the characterization of the nanoparticles in situ but also the observation of a thermal phase transition. Critically, because these assemblies form from thermoresponsive components, any temperature changes during sample preparation and analysis can alter morphologies, necessitating direct in situ characterization of reaction progression. We believe that the findings and the technique described herein will lead to unprecedented possibilities for the development and characterization of self-organizing soft matter.

An in-situ TEM microreactor for real-time nanomorphology & physicochemical parameters interrelated characterization

In-situ quantitative structure-property relationship (QSPR) characterization between nanomorphology and physicochemical parameters is essential for revealing the growth mechanism and guiding the optimization of functional nanomaterials. However, extracting the interrelated physicochemical parameters in the process of nanomorphology observation is still challenging. Herein, this paper demonstrates an advanced chip-scale platform for in-situ QSPR characterization by integrating a gravimetric resonant microcantilever sensor within a transmission electron microscopy (TEM) microreactor. The microcantilever measures the thermodynamic and kinetic parameters, such as enthalpy (ΔH°), activation energy (Ea) and reaction rate (v), by monitoring the reaction-induced mass change of functional nanomaterials. Simultaneously, the low-stress SiNx films as electron transparent windows allow in-situ nanomorphology observation. Using such a QSPR characterization platform, the three-stage sulfuration mechanism based on nano Kirkendall effect for the gas-solid reaction of H2S and Cu nanowires is revealed, and the different molecule-interaction behaviors of SO2 on ZnO nanowires at various temperatures are distinguished for the first time. It is believed that the in-situ QSPR microreactor can be widely used for elucidating the underlying interfacial thermodynamic/kinetic reaction mechanism, and guiding the controllable synthesis and precise optimization of functional nanomaterials.

Automated cryo-lamella preparation for high-throughput in-situ structural biology

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200–300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Imaging Electrochemistry at the Nanoscale By Liquid Cell Transmission Electron Microscopy

Direct optical visualization of electrochemical processes at the nanoscale is an exciting field of research. It became more and more feasible through the development of enclosed liquid cells for transmission electron microscopes over the past years [1]. The liquid cell itself is built by stacking two silicon chips with electron transparent silicon nitride windows, which are then sealed by a gasket against the surrounding high vacuum in the microscope column. Microfabrication of electrode structures onto the chips opened up the opportunity to control the electric potential and current within the cell in order to perform in-situ potentiometric measurements and impedance spectroscopy. This unique feature enables the correlation of real-time optical imaging at the nanoscale and electrochemistry. So far, most of the experiments have focused on batteries and corrosion studies [2,3]. While there have been numerous reports on the diffusion and agglomeration of nanoparticles [4], the visualization of nanoparticle dynamics coupled to voltammetry in the vicinity of an electrode surface has received less attention. Herein, we investigate the diffusion, deposition and stripping of nanoparticles at an electrode under an applied electric bias. We give insight into how a nanoparticle collision event quantitatively transfers into an electrochemical current signal and disentangle electron beam effects. These results might pave the way towards observation and understanding of reactions catalyzed by nanoparticles in general.

In Aqua Electrochemistry Probed by XPEEM: Experimental Setup, Examples, and Challenges

Recent developments in environmental and liquid cells equipped with electron transparent graphene windows have enabled traditional surface science spectromicroscopy tools, such as scanning X-ray photoelectron microscopy, X-ray photoemission electron microscopy (XPEEM), and scanning electron microscopy to be applied for studying solid–liquid and liquid–gas interfaces. Here, we focus on the experimental implementation of XPEEM to probe electrified graphene–liquid interfaces using electrolyte-filled microchannel arrays as a new sample platform. We demonstrate the important methodological advantage of these multi-sample arrays: they combine the wide field of view hyperspectral imaging capabilities from XPEEM with the use of powerful data mining algorithms to reveal spectroscopic and temporal behaviors at the level of the individual microsample or the entire array ensemble.

Through-membrane electron-beam lithography for ultrathin membrane applications

We present a technique to fabricate ultrathin (down to 20 nm) uniform electron transparent windows at dedicated locations in a SiN membrane for in situ transmission electron microscopy experiments. An electron-beam (e-beam) resist is spray-coated on the backside of the membrane in a KOH-etched cavity in silicon which is patterned using through-membrane electron-beam lithography. This is a controlled way to make transparent windows in membranes, whilst the topside of the membrane remains undamaged and retains its flatness. Our approach was optimized for MEMS-based heating chips but can be applied to any chip design. We show two different applications of this technique for (1) fabrication of a nanogap electrode by means of electromigration in thin free-standing metal films and (2) making low-noise graphene nanopore devices.

Note on in situ (scanning) transmission electron microscopy study of liquid samples

Liquid cell (scanning) transmission electron microscopy has been developed rapidly, using amorphous SiNx membranes as electron transparent windows. The current interpretations of electron beam effects are mainly based on radiolytic processes. In this note, additional effects of the electric field due to electron-beam irradiation are discussed. The electric field can be produced by the charge accumulation due to the emission of secondary and Auger electrons. Besides various beam-induced phenomena, such as nanoparticle precipitation and gas bubble formation and motion, two other effects need to be considered; one is the change of Gibbs free energy of nucleation and the other is the violation of Brownian motion due to ion drifting driven by the electric field.

MEMS‐based nanoreactor for in situ analysis of solid–gas interactions inside the transmission electron microscope

A nanoreactor-based system for in situ analysis of solid–gas interactions inside the transmission electron microscope, referred to as the ‘climate system’, is introduced here. The latter uses an micro-electro mechanical systems (MEMS)-based device as a multi-functional sample carrier and microsized laboratory for simultaneous heating and gas experiments. To assemble the nanoreactor, two chips are sandwiched together to form a minimised chemical reaction chamber. The bottom chip contains a four-point-probe microheater, which controls and introduces the heating environment (up to 1300°C) around the sample. Similarly, it contains the spacers that define the height of the gas chamber. The top chip confines the gas in the microchamber while isolating it from the external environment. Both chips contain electron transparent windows that allow the electron beam to pass through for in situ imaging. The sample can be deposited or prepared directly on the windows, which are made of silicon nitride and can sustain pressures up to 1.5 bar and high temperatures while ensuring low background atomic resolution imaging.

Atomically Thin Graphene Windows That Enable High Contrast Electron Microscopy without a Specimen Vacuum Chamber.

Scanning electron microscopes (SEMs) require a high vacuum environment to generate and shape an electron beam for imaging; however, the vacuum conditions greatly limit the nature of specimens that can be examined. From a purely scattering physics perspective, it is not necessary to place the specimen inside the vacuum chamber-the mean free paths (MFPs) for electron scattering in air at typical SEM beam voltages are 50-100 μm. This is the idea behind the airSEM, which removes the specimen vacuum chamber from the SEM and places the sample in air. The thickness of the gas layer is less than a MFP from an electron-transparent window to preserve the shape and resolution of the incident beam, resulting in comparable imaging quality to an all-vacuum SEM. Present silicon nitride windows scatter far more strongly than the air gap and are currently the contrast and resolution limiting factor in the airSEM. Graphene windows have been used previously to wrap or seal samples in vacuum for imaging. Here we demonstrate the use of a robust bilayer graphene window for sealing the electron optics from the room environment, providing an electron transparent window with only a 2% drop in contrast. There is a 5-fold-increase in signal/noise ratio for imaging compared to multi-MFP-thick silicon nitride windows, enabling high contrast in backscattered, transmission, and surface imaging modes for the new airSEM geometry.

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber

A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope's objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Monte Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400 μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens and in situ chemical and electrochemical processes.

Assessing Photocatalytic Activity at the Nanoscale Using Integrated Optical and Electron Microscopy

An integrated optical‐electron microscope is presented that enables the in situ study of dynamic processes on photoactive materials. Here, the deposition of metallic silver nanostructures at ZnO photocatalyst particles is monitored in real time under ambient conditions by means of scanning electron microscopy. Zinc oxide crystals are immobilized on an electron transparent silicon nitride window. By passing UV light through an opposing optically transparent window, the zinc oxide is illuminated resulting in the photocatalytic formation of silver nanostructures. Both windows are part of a specially designed liquid cell filled with a dilute aqueous silver nitrate solution. Using the presented system, different electron detectors are evaluated for their ability to provide detailed images despite the interference caused by the liquid surrounding the sample. Special care has to be taken since direct silver reduction from solution induced by the electron beam interferes with the photocatalytic process. Oxygen gas, produced during the photocatalytic reaction, is also shown to complicate the imaging of the dynamic nanoscale processes in the scanning electron microscope. Nevertheless, the integrated approach allows to directly establish structure–activity relationships and to unravel optically induced processes at nanostructured materials.

In Situ Ptychography of Heterogeneous Catalysts using Hard X-Rays: High Resolution Imaging at Ambient Pressure and Elevated Temperature.

A new closed cell is presented for in situ X-ray ptychography which allows studies under gas flow and at elevated temperature. In order to gain complementary information by transmission and scanning electron microscopy, the cell makes use of a Protochips E-chipTM which contains a small, thin electron transparent window and allows heating. Two gold-based systems, 50 nm gold particles and nanoporous gold as a relevant catalyst sample, were used for studying the feasibility of the cell. Measurements showing a resolution around 40 nm have been achieved under a flow of synthetic air and during heating up to temperatures of 933 K. An elevated temperature exhibited little influence on image quality and resolution. With this study, the potential of in situ hard X-ray ptychography for investigating annealing processes of real catalyst samples is demonstrated. Furthermore, the possibility to use the same sample holder for ex situ electron microscopy before and after the in situ study underlines the unique possibilities available with this combination of electron microscopy and X-ray microscopy on the same sample.

Fabrication of high quality plan-view TEM specimens using the focused ion beam

We describe a technique using a focused ion beam instrument to fabricate high quality plan-view specimens for transmission electron microscopy studies. The technique is simple, site-specific and is capable of fabricating multiple large, >100μm2 electron transparent windows within epitaxially grown thin films. A film of La0.67Sr0.33MnO3 is used to demonstrate the technique and its structural and functional properties are surveyed by high resolution imaging, electron spectroscopy, atomic force microscopy and Lorentz electron microscopy. The window is demonstrated to have good thickness uniformity and a low defect density that does not impair the film's Curie temperature. The technique will enable the study of in-plane structural and functional properties of a variety of epitaxial thin film systems.

Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography

Cryo-electron tomography (CET) is ideally suited for bridging the resolution gap between molecular and cellular structural studies. However, CET is limited to a sample thickness under 500nm, which is thinner than most cells. Here, we review a method for preparing cells for CET using focused-ion-beam milling, a technique commonly used in materials science. Adapted to cryogenic conditions, FIB milling can be applied to various cell types to produce samples thin enough for CET that do not present the artefacts typical to other preparation techniques, for example, cryo-ultramicrotomy, effectively opening windows into intact cells. Samples can be produced routinely and reproducibly. The data obtained from CET can be used for structural studies in situ, or to do quantitative cell biology studies, in which cells can be observed at the molecular level under different physiological conditions.

In-situ SEM microchip setup for electrochemical experiments with water based solutions

Studying electrochemical (EC) processes with electron microscopes offers the possibility of achieving much higher resolution imaging of nanoscale processes in real time than with optical microscopes. We have developed a vacuum sealed liquid sample electrochemical cell with electron transparent windows, microelectrodes and an electrochemical reference electrode. The system, called the EC-SEM Cell, is used to study electrochemical reactions in liquid with a standard scanning electron microscope (SEM). The central component is a microfabricated chip with a thin (50 nm) Si-rich silicon nitride (SiNx) window with lithographically defined platinum microelectrodes. We show here the design principles of the EC-SEM system, its detailed construction and how it has been used to perform a range of EC experiments, two of which are presented here. It is shown that the EC-SEM Cell can survive extended in-situ EC experiments. Before the EC experiments we characterized the beam current being deposited in the liquid as this will affect the experiments. The first EC experiment shows the influence of the electron-beam (e-beam) on a nickel solution by inducing electroless nickel deposition on the window when increasing the current density from the e-beam. The second experiment shows electrolysis in EC-SEM Cell, induced by the built-in electrodes.

Characterization of LPCVD amorphous silicon carbide (a-SiC) as material for electron transparent windows

We investigate on the physical and chemical properties of non-stoichiometric amorphous silicon carbide (a-SiCx) thin layers deposited by low-pressure chemical vapor deposition (LPCVD). Characterizations are specifically tailored to understand the suitability of a-SiCx as electron transparent window material. The structure, composition, continuity, intrinsic stress, etch-rates and roughness of the layers are determined. The a-SiCx layers are highly uniform and continuous, showing tensile intrinsic stress and high chemical inertness. The layer with the lowest roughness (0.22 nm) and intrinsic stress (0.7 GPa) is employed to fabricate electron transparent windows of 16 nm for use in a Transmission Electron Microscope (TEM). The resulting windows are highly transparent, enabling the acquisition of TEM images down to the resolution limit of the microscope (0.12 nm). Moreover, the a-SiCx windows show a resistance to electron beam damage which is up to 9 times higher than that of the commonly employed low-stress LPCVD silicon nitride (SiNx). Therefore, the proposed a-SiCx is particularly suited for TEM experiments where high chemical inertness and/or long exposures to the TEM electron beam are required. In addition, the a-SiCx layer could advantageously be employed in the design of microelectromechanical systems (MEMS), especially those operating in harsh environments.

Electron transparent graphene windows for environmental scanning electron microscopy in liquids and dense gases

Due to its ultrahigh electron transmissivity in a wide electron energy range, molecular impermeability, high electrical conductivity and excellent mechanical stiffness, suspended graphene membranes appear to be a nearly ideal window material for in situ (in vivo) environmental electron microscopy of nano- and mesoscopic objects (including bio-medical samples) immersed in liquids and/or in dense gaseous media. In this paper, taking advantage of a small modification of the graphene transfer protocol onto metallic and SiN supporting orifices, reusable environmental cells with exchangeable graphene windows have been designed. Using colloidal gold nanoparticles (50 nm) dispersed in water as model objects for scanning electron microscopy in liquids as proof of concept, different conditions for imaging through the graphene membrane were tested. Limiting factors for electron microscopy in liquids, such as electron beam induced water radiolysis and damage of the graphene membrane at high electron doses, are discussed.

Charged nanoparticle dynamics in water induced by scanning transmission electron microscopy.

Using scanning transmission electron microscopy we image ~4 nm platinum nanoparticles deposited on an insulating membrane, where the membrane is one of two electron-transparent windows separating an aqueous environment from the microscope's high vacuum. Upon receiving a relatively moderate dose of ~10(4) e/nm(2), initially immobile nanoparticles begin to move along trajectories that are directed radially outward from the center of the field of view. With larger dose rates the particle motion becomes increasingly dramatic. These observations demonstrate that, even under mild imaging conditions, the in situ electron microscopy of aqueous environments can produce electrophoretic charging effects that dominate the dynamics of nanoparticles under observation.

Video-frequency scanning transmission electron microscopy of moving gold nanoparticles in liquid

Immobilized gold nanoparticles were imaged in a liquid containing water and 50% glycerol with scanning transmission electron microscopy (STEM). The specimen was enclosed in a liquid compartment formed by two silicon microchips with electron transparent windows. A series of images was recorded at video frequency with a spatial resolution of 1.5nm. The nanoparticles detached from their support after imaging them for several seconds at a magnification of 250,000. Their movement was found to be much different than the movement of nanoparticles moving freely in liquid as described by Brownian Motion. The direction of motion was not random—the nanoparticles moved either in a preferred direction, or radially outwards from the center of the image. The displacement of the gold nanoparticles over time was three orders of magnitude smaller than expected on the basis of Brownian Motion. This finding implies that nanoscale objects of flexible structure or freely floating, including nanoparticles and biological objects, can be imaged with nanoscale resolution, as long as they are in close proximity to a solid support structure.

In-situ TEM on (de)hydrogenation of Pd at 0.5–4.5 bar hydrogen pressure and 20–400°C

We have developed a nanoreactor, sample holder and gas system for in-situ transmission electron microscopy (TEM) of hydrogen storage materials up to at least 4.5 bar. The MEMS-based nanoreactor has a microheater, two electron-transparent windows and a gas inlet and outlet. The holder contains various O-rings to have leak-tight connections with the nanoreactor. The system was tested with the (de)hydrogenation of Pd at pressures up to 4.5 bar. The Pd film consisted of islands being 15 nm thick and 50-500 nm wide. In electron diffraction mode we observed reproducibly a crystal lattice expansion and shrinkage owing to hydrogenation and dehydrogenation, respectively. In selected-area electron diffraction and bright/dark-field modes the (de)hydrogenation of individual Pd particles was followed. Some Pd islands are consistently hydrogenated faster than others. When thermally cycled, thermal hysteresis of about 10-16°C between hydrogen absorption and desorption was observed for hydrogen pressures of 0.5-4.5 bar. Experiments at 0.8 bar and 3.2 bar showed that the (de)hydrogenation temperature is not affected by the electron beam. This result shows that this is a fast method to investigate hydrogen storage materials with information at the nanometer scale.