Time-resolved Electron Microscopy Research Articles

Mechanisms of corrosive freeze-thaw damage in AA7075 using time-resolved x-ray microtomography and correlative electron microscopy

Aluminum aircraft structures experience severe corrosion from exposure to aggressive chloride environments, including cyclic freezing and thawing of residual water during ascent and descent, introducing a cyclic freeze-thaw component to the corrosion process. While corrosion mechanisms in aircraft structures are well studied at constant temperatures, the microstructural and mechanistic behavior under freeze-and-thaw conditions remains unclear. To understand transformations induced by cyclic temperature, we used three-dimensional (3D) x-ray computed tomography (XCT) with scanning electron microscopy (SEM) to study the behavior of AA7075-T651 in a simulated seawater environment undergoing freezing and thawing cycles. Rods immersed in saltwater were thermally cycled above and below freezing, and structural changes were intermittently characterized in 3D. Under freeze-thaw conditions, cracks initiated within corrosion pits through ice expansion, causing progressive crevice growth and spalling along inclusions and grain boundaries with intermediate misorientation angles. Damage mechanisms in freeze-thaw and conventional corrosion environments are compared, with correlations to microstructural evolution.

Ultrastructural membrane dynamics of mouse and human cortical synapses.

Live human brain tissues provide unique opportunities for understanding the physiology and pathophysiology of synaptic transmission. Investigations have been limited to anatomy, electrophysiology, and protein localization-while crucial parameters such as synaptic vesicle dynamics were not visualized. Here we utilize zap-and-freeze time-resolved electron microscopy to overcome this hurdle. First we validate the approach with acute mouse brain slices to demonstrate that axons parallel to the electrical field can be stimulated to produce calcium signaling. Next we show that ultrafast endocytosis is induced and can be captured in both mouse and human brain slices. Crucially, in both species a protein essential for ultrafast endocytosis Dynamin 1xA (Dyn1xA) localizes to the region peripheral to the active zone, the putative endocytic zone, indicating a likely mechanism conservation between mouse and human. This approach has the potential to reveal dynamic, high-resolution information about synaptic membrane trafficking in intact human brain slices.

Laser-Induced Stress-Driven Nanoplate Jumping Visualized by Ultrafast Electron Microscopy.

Understanding laser-induced jumping has attracted great interest in nanomaterial launching and transfer but requires a high spatiotemporal resolution visualization. Here, we report a jumping dynamics of nanoplate driven by ultrafast laser-induced stress using time-resolved transmission electron microscopy. Single-shot imaging reveals a nondestructive launching of gold nanoplates in several nanoseconds after the pulsed femtosecond laser excitation. The temperature rise and acoustic vibration, derived from ultrafast electron crystallography with a picosecond time resolution, confirm the existence of a laser-induced elastic stress wave. The generation, propagation, and reflection of thermal stress waves are further clarified by atomic simulation. The nonequilibrium ultrafast laser heating produces a compressive stress wave within several picoseconds, constrained by the supporting substrate under nanoplate to provide thrust force. This compressive stress is subsequently reflected into tensile stress by the substrate, promoting the nanoplate to jump off the substrate. Furthermore, the uneven interface adhesion results in the jumping flip of nanoplates, as well as, diminished their jumping speed. This study unveils the jumping regime driven by impulsive laser-excited stress and offers understanding of light-matter interaction.

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

Attosecond electron microscopy by free-electron homodyne detection

Time-resolved electron microscopy aims to track nanoscale excitations and dynamic states of matter at a temporal resolution ultimately reaching the attosecond regime. Periodically time-varying fields in an illuminated specimen cause free-electron inelastic scattering, which enables the spectroscopic imaging of near-field intensities. However, access to the evolution of nanoscale fields and structures within the cycle of light requires sensitivity to the optical phase. Here we introduce free-electron homodyne detection as a universally applicable approach to electron microscopy of phase-resolved optical responses at high spatiotemporal resolution. In this scheme, a phase-controlled reference interaction serves as the local oscillator to extract arbitrary sample-induced modulations of a free-electron wavefunction. We demonstrate this principle through the phase-resolved imaging of plasmonic fields with few-nanometre spatial and sub-cycle temporal resolutions. Due to its sensitivity to both phase- and amplitude-modulated electron beams, free-electron homodyne detection measurements will be able to detect and amplify weak signals stemming from a wide variety of microscopic origins, including linear and nonlinear optical polarizations, atomic and molecular resonances, and attosecond-modulated structure factors.

Atomic-Scale Time-Resolved Imaging of Krypton Dimers, Chains and Transition to a One-Dimensional Gas.

Single-atom dynamics of noble-gas elements have been investigated using time-resolved transmission electron microscopy (TEM), with direct observation providing for a deeper understanding of chemical bonding, reactivity, and states of matter at the nanoscale. We report on a nanoscale system consisting of endohedral fullerenes encapsulated within single-walled carbon nanotubes ((Kr@C60)@SWCNT), capable of the delivery and release of krypton atoms on-demand, via coalescence of host fullerene cages under the action of the electron beam (in situ) or heat (ex situ). The state and dynamics of Kr atoms were investigated by energy dispersive X-ray spectroscopy (EDS), electron energy loss spectroscopy (EELS), and X-ray photoelectron spectroscopy (XPS). Kr atom positions were measured precisely using aberration-corrected high-resolution TEM (AC-HRTEM), aberration-corrected scanning TEM (AC-STEM), and single-atom spectroscopic imaging (STEM-EELS). The electron beam drove the formation of 2Kr@C120 capsules, in which van der Waals Kr2 and transient covalent [Kr2]+ bonding states were identified. Thermal coalescence led to the formation of longer coalesced nested nanotubes containing more loosely bound Krn chains (n = 3-6). In some instances, delocalization of Kr atomic positions was confirmed by STEM analysis as the transition to a one-dimensional (1D) gas, as Kr atoms were constrained to only one degree of translational freedom within long, well-annealed, nested nanotubes. Such nested nanotube structures were investigated by Raman spectroscopy. This material represents a highly compressed and dimensionally constrained 1D gas stable under ambient conditions. Direct atomic-scale imaging has revealed elusive bonding states and a previously unseen 1D gaseous state of matter of this noble gas element, demonstrating TEM to be a powerful tool in the discovery of chemistry at the single-atom level.

CryoEM reveals the complex self-assembly of a chemically driven disulfide hydrogel.

Inspired by the adaptability of biological materials, a variety of synthetic, chemically driven self-assembly processes have been developed that result in the transient formation of supramolecular structures. These structures form through two simultaneous reactions, forward and backward, which generate and consume a molecule that undergoes self-assembly. The dynamics of these assembly processes have been shown to differ from conventional thermodynamically stable molecular assemblies. However, the evolution of nanoscale morphologies in chemically driven self-assembly and how they compare to conventional assemblies has not been resolved. Here, we use a chemically driven redox system to separately carry out the forward and backward reactions. We analyze the forward and backward reactions both sequentially and synchronously with time-resolved cryogenic transmission electron microscopy (cryoEM). Quantitative image analysis shows that the synchronous process is more complex and heterogeneous than the sequential process. Our key finding is that a thermodynamically unstable stacked nanorod phase, briefly observed in the backward reaction, is sustained for ∼6 hours in the synchronous process. Kinetic Monte Carlo modeling show that the synchronous process is driven by multiple cycles of assembly and disassembly. The collective data suggest that chemically driven self-assembly can create sustained morphologies not seen in thermodynamically stable assemblies by kinetically stabilizing transient intermediates. This finding provides plausible design principles to develop and optimize supramolecular materials with novel properties.

Disease-specific tau filaments assemble via polymorphic intermediates

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297–391) into paired helical filaments of Alzheimer’s disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302–316. Nuclear magnetic resonance indicates that the same residues adopt rigid, β-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

New Insights into Internal Oxidation of Alloy 690 and Model Alloys in Hydrogenated Steam

The oxidation behavior of Alloy 690 and Ni-xCr-10Fe model alloys exposed to 480°C hydrogenated steam, in the nickel metal stability region, was studied using a variety of techniques, including time-resolved scanning electron microscopy, transmission electron microscopy, time-of-flight secondary ion mass spectrometry, and electron energy loss spectroscopy. The alloy underwent internal oxidation intragranularly, resulting in the expulsion of metallic Ni nodules to the surface. A compact Cr-rich “healing” layer developed over time at the intragranular internal oxidation front near grain boundaries and significantly retards further internal oxidation. Protective external Cr-rich oxide formed at and very near the grain boundaries, hindering intergranular oxidation. In view of the fact that the protective oxide forms micrometers away from the grain boundaries, one cannot simply say that Cr diffuses up the boundary and forms an oxide that spreads laterally—internal stress relief must play a role, just as it does within the grains, only with different mechanisms at play.

Cinematographic study ofstochastic chemical events at atomic resolution.

The advent of single-molecule atomic-resolution time-resolved electron microscopy (SMART-EM) has created a new field of 'cinematic chemistry,' allowing for the cinematographic recording of dynamic behaviors of organic and inorganic molecules and their assembly. However, the limited electron dose per frame of video images presents a major challenge in SMART-EM. Recent advances in direct electron counting cameras and techniques to enhance image quality through the implementation of a denoising algorithm have enabled the tracking of stochastic molecular motions and chemical reactions with sub-millisecond temporal resolution and sub-angstrom localization precision. This review showcases the development of dynamic molecular imaging using the SMART-EM technique, highlighting insights into nanomechanical behavior during molecular shuttle motion, pathways of multistep chemical reactions, and elucidation of crystallization processes at the atomic level.

Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C).

Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.

Time-resolved imaging and analysis of the electron beam-induced formation of an open-cage metallo-azafullerene.

The visualization of single-molecule reactions provides crucial insights into chemical processes, and the ability to do so has grown with the advances in high-resolution transmission electron microscopy. There is currently a limited mechanistic understanding of chemical reactions under the electron beam. However, such reactions may enable synthetic methodologies that cannot be accessed by traditional organic chemistry methods. Here we demonstrate the synthetic use of the electron beam, by in-depth single-molecule, atomic-resolution, time-resolved transmission electron microscopy studies, in inducing the formation of a doubly holed fullerene-porphyrin cage structure from a well-defined benzoporphyrin precursor deposited on graphene. Through real-time imaging, we analyse the hybrid's ability to host up to two Pb atoms, and subsequently probe the dynamics of the Pb-Pb binding motif in this exotic metallo-organic cage structure. Through simulation, we conclude that the secondary electrons, which accumulate in the periphery of the irradiated area, can also initiate chemical reactions. Consequently, designing advanced carbon nanostructures by electron-beam lithography will depend on the understanding and limitations of molecular radiation chemistry.

Understanding the Chemical Mechanism behind Photoinduced Enhanced Raman Spectroscopy

Photoinduced enhanced Raman spectroscopy (PIERS) is a new surface enhanced Raman spectroscopy (SERS) modality with a 680% Raman signal enhancement of adsorbed analytes over that of SERS. Despite the explosion in recent demonstrations, the PIERS mechanism remains undetermined. Using X-ray and time-resolved optical spectroscopies, electron microscopy, cyclic voltammetry, and density functional theory simulations, we elucidate the atomic-scale mechanism behind PIERS. Stable PIERS substrates were fabricated using self-organized arrays of TiO2 nanotubes with controlled oxygen vacancy doping and size-controlled silver nanoparticles. The key source of PIERS vs SERS enhancement is an increase in the Raman polarizability of the adsorbed analyte upon photoinduced charge transfer. A balance between improved crystallinity, which enhances charge transfer due to higher electron mobility but decreases light absorption, and increased oxygen vacancy defect concentration, which increases light absorption, is critical. This work enables the rational design of PIERS substrates for sensing.

A simple flash and freeze system for cryogenic time-resolved electron microscopy.

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.

Time-resolved transmission electron microscopy for nanoscale chemical dynamics.

The ability of transmission electron microscopy (TEM) to image a structure ranging from millimetres to Ångströms has made it an indispensable component of the toolkit of modern chemists. TEM has enabled unprecedented understanding of the atomic structures of materials and how structure relates to properties and functions. Recent developments in TEM have advanced the technique beyond static material characterization to probing structural evolution on the nanoscale in real time. Accompanying advances in data collection have pushed the temporal resolution into the microsecond regime with the use of direct-electron detectors and down to the femtosecond regime with pump-probe microscopy. Consequently, studies have deftly applied TEM for understanding nanoscale dynamics, often in operando. In this Review, time-resolved in situ TEM techniques and their applications for probing chemical and physical processes are discussed, along with emerging directions in the TEM field.

Tracking the Growth of Chiral Plasmonic Nanocrystals at Molybdenum Disulfide Heterostructural Interfaces

The way of accurately regulating the growth of chiral plasmonics is of great importance for exploring the chirality information and improving its potential values. Herein, cysteine enantiomers modulate the anisotropic and epitaxial growth of gold nanoplasmonics on seeds of exfoliated MoS2 nanosheets. The heterostructural Au and MoS2 hybrids induced by enantiomeric cysteine are presented with chiroptical characteristics, dendritic morphologies, and plasmonic performances. Moreover, the synthesis, condition optimization, formation mechanism, and plasmonic properties of Au and MoS2 dendritic nanostructures are studied. The chirality characteristics are identified using the circular dichroism spectra and scanning electron microscopy. Time-resolved transmission electron microscopy and UV-vis spectra of the intermediate products captured are analyzed to confirm the formation mechanism of dendritic plasmonic nanostructures at heterostructural surfaces. The specific dendritic morphologies originate from the synergistic impacts of heterostructural MoS2 interfaces and enantiomeric cysteine-induced anisotropic manipulation. Significantly, the developed synthesis strategy of chiral nanostructures at heterostructural interfaces is highly promising in promoting the understanding of the plasmonic function and crucial chirality bioinformation.

Direct measurement of single-molecule dynamics and reaction kinetics in confinement using time-resolved transmission electron microscopy.

We report experimental methodologies utilising transmission electron microscopy (TEM) as an imaging tool for reaction kinetics at the single molecule level, in direct space and with spatiotemporal continuity. Using reactions of perchlorocoronene (PCC) in nanotubes of different diameters and at different temperatures, we found a period of molecular movement to precede the intermolecular addition of PCC, with a stronger dependence of the reaction rate on the nanotube diameter, controlling the local environments around molecules, than on the reaction temperature (-175, 23 or 400 °C). Once initiated, polymerisation of PCC follows zero-order reaction kinetics with the observed reaction cross section σobs of 1.13 × 10-9 nm2 (11.3 ± 0.6 barn), determined directly from time-resolved TEM image series acquired with a rate of 100 frames per second. Polymerisation was shown to proceed from a single point, with molecules reacting sequentially, as in a domino effect, due to the strict conformational requirement of the Diels-Alder cycloaddition creating the bottleneck for the reaction. The reaction mechanism was corroborated by correlating structures of reaction intermediates observed in TEM images, with molecular weights measured by using mass spectrometry (MS) when the same reaction was triggered by UV irradiation. The approaches developed in this study bring the imaging of chemical reactions at the single-molecule level closer to traditional concepts of chemistry.

Support-facet-dependent morphology of small Pt particles on ceria.

Direct atomic scale information on how the structure of supported nanoparticles is affected by the metal-support interaction is rare. Using scanning transmission electron microscopy, we provide direct evidence of a facet-dependent support interaction for Pt nanoparticles on CeO2, governing the dimensionality of small platinum particles. Our findings indicate that particles consisting of less than ∼130 atoms prefer a 3D shape on CeO2(111) facets, while 2D raft structures are favored on CeO2(100) facets. Measurements of stationary particles on both surface facets are supplemented by time resolved measurements following a single particle with atomic resolution as it migrates from CeO2(111) to CeO2(100), undergoing a dimensionality change from 3D to 2D. The intricate transformation mechanism reveals how the 3D particle disassembles and completely wets a neighboring CeO2(100) facet. Density functional theory calculations confirm the structure-trend and reveal the thermodynamic driving force for the migration of small particles. Knowledge of the presented metal-support interactions is crucial to establish structure-function relationships in a range of applications based on supported nanostructures.

Temporal resolution in transmission electron microscopy using a photoemission electron source.

Temporal resolution in transmission electron microscopy (TEM) has progressed to the sub-picosecond level with the stroboscopic method using a photoemission mechanism with an ultrafast laser for the electron gun. Time-resolved TEM in conjunction with a photocathode (PC)-type electron source pumped by a pulsed laser has been actively developed to exceed sub-nanosecond time resolution. Here, we provide an overview of the trends in this field and discuss the measurement targets that can be obtained by time-resolved measurements. Furthermore, we consider the types and characteristics of PC materials and their related physical quantities for evaluation of electron beam properties. Experimental results obtained by time-resolved TEM using a semiconductor PC that has a surface with a negative electron affinity are presented, and application results based on quantum mechanics are given. We also describe new techniques for improving the time resolution and new applications of pulsed electron beams in electron microscopy and discuss the measurement targets that are expected for time-resolved electron microscopy.

Transient electron energy-loss spectroscopy of optically stimulated gold nanoparticles using picosecond pulsed electron beam

Ultrafast phenomena in gold nanotriangles (AuNTs) were investigated using a transient electron energy-loss spectroscopy (TEELS) technique under irradiation from a 150-fs pulse laser with a wavelength of 780 nm. This investigation was conducted using a time-resolved transmission electron microscopy method that was developed to measure the dynamics of nanomaterials. Enhancement of the intensity and energy-width broadening of the energy loss were observed at the EEL peaks associated with surface and bulk plasmons on the AuNTs. The TEELS measurement revealed two decay processes of 7.8 ps and longer than 100 ps that compensate for relaxation times of excited surface plasmons using transient absorption spectroscopy. The results show that the bulk and surface plasmons have the same time evolution, i.e., that the excited electrons on the surface and in the bulk have the same relaxation processes in both electron–phonon and phonon–phonon interactions. The time evolution of electronic and lattice temperatures was also estimated based on the measured relaxation time using a two-temperature model, which revealed the volume expansion of the AuNTs and clarified the energy shifts of plasmons. Details of excited electrons in nanoparticles investigated via plasmon energy loss are expected to facilitate improvement in the performance for energy harvesting of photons in nanostructure-controlled materials.