Conventional Transmission Electron Microscopy Research Articles

Quantification of nanoscale precipitation in AA7050 using X-ray scattering, electron microscopy and automated particle counting techniques

Material strength is dependent on several factors, including precipitation strengthening, which is the main strengthening mechanism in AA7050 aluminum alloys. These alloys contain numerous nanometer-scale precipitates that occur throughout grains and along grain boundaries when subjected to a range of heat treatment conditions. In this study, three different characterization methods were used to characterize these nanoscale precipitates: conventional scanning transmission electron microscopy (STEM); laboratory-based small-angle X-ray scattering (SAXS); and a new software analysis tool developed by Thermo Fisher Scientific: automated particle workflow (APW). Each method was used to determine average precipitate size and volume fraction of precipitation in AA7050-T7451 specimens with variable post-T7 heat treatment procedures. STEM techniques measured the average particle size as ranging from 7.6 to 17.6 nm, compared to 6.3–10.7 nm for SAXS, and 7.4–10.8 nm for APW. Volume fraction determinations varied from 0.23 to 16 %, depending on method, and were the most difficult to quantify. Significant outcomes of the current study are that SAXS and APW are the most accurate methods for determining average particle size and that future work is needed to effectively analyze precipitate volume fraction.

Cyto-architecture of Byblis glands and leaf cells based on freeze-substitution and conventional TEM.

Byblis liniflora (Byblidaceae) is a carnivorous plant that has developed sticky fly paper traps with two types of glandular trichomes producing digestive enzymes and sticky mucilage. This study aimed to analyze the ultrastructure of these glandular leaf trichomes based on rapid freeze fixation and conventional chemical fixation in the attempt to understand their functional contribution to the carnivorous performance of the plants. The Byblis cells were studied in TEM, SEM and STEM using cryo techniques for fixation and substitution in addition to conventional chemical fixation. We show in detail the architecture of both the digestive glands and the mucilage glands with their relevant sets of organelles. Both mitochondria and plastids have a conspicuous plasticity, with branches and constrictions, and they associate to form clusters. The glandular cells appear to be transfer cells with cell wall ingrowths. Digestive glands occur in different states of development. Their cuticle forms discontinuities which are unique among glands of carnivorous plants. They look like cuticular holes -- the cuticle separates from the cell wall in only one spot and then ruptures. Cuticular discontinuities thus differ from cuticular gaps and cuticular pores so far described in carnivorous plants. We therefore propose for them the term cuticular holes. Application of cryo-techniques made it possible to show the true structure of the cell wall and the relationship between cell wall ingrowths and organelles, as well as the morphology and structure of organelles and their associations.

Structures and migrations of interfaces between β precipitates and α′ matrix in a Ti-2.6 wt% Mo alloy

While the precipitation of α (hcp) from β (bcc) in various Ti alloys has been investigated in great detail, less is known about the precipitation of β from α. This study conducts a systematic investigation of structures and migrations of interfaces between β precipitates and α′ matrix in a Ti-2.6 wt% Mo alloy. Utilizing conventional and high-resolution transmission electron microscopy (TEM), assisted with molecular dynamics and generalized O-element analysis approaches, we unveil the dislocation and atomic structures of three typical portions of the interface surrounding a β precipitate, i.e., the habit plane, the side facet, and the end face. The habit plane of β precipitates contains two sets of dislocations with Burgers vectors b1=[21¯1¯3]α/6|[11¯1]β/2 and b4=[21¯1¯3¯]α/6|[111¯]β/2, in contrast to the single set of b1 dislocations previously found on the habit plane of α laths precipitated from β matrix in various Ti alloys. Interface migrations during shrinkage of β precipitates are characterized via in-situ TEM, showing that both the habit plane and side facet migrate through the lateral motion of nanometer-high growth ledges. The dislocation structures of various growth ledges were obtained through generalized O-element calculations, suggesting a potential non-conservative dislocation trajectory as semicoherent growth ledges sweep across the interface.

Unsupervised machine learning and cepstral analysis with 4D-STEM for characterizing complex microstructures of metallic alloys

Four-dimensional scanning transmission electron microscopy, coupled with a wide array of data analytics, has unveiled new insights into complex materials. Here, we introduce a straightforward unsupervised machine learning approach that entails dimensionality reduction and clustering with minimal hyperparameter tuning to semi-automatically identify unique coexisting structures in metallic alloys. Applying cepstral transformation to the original diffraction dataset improves this process by effectively isolating phase information from potential signal ambiguity caused by sample tilt and thickness variations, commonly observed in electron diffraction patterns. In a case study of a NiTiHfAl shape memory alloy, conventional scanning transmission electron microscopy imaging struggles to accurately identify a low-contrast precipitate at lower magnifications, posing challenges for microscale analyses. We find that our method efficiently separates multiple coherent structures while using objective means of determining hyperparameters. Furthermore, we demonstrate how the clustering result facilitates more robust strain mapping to provide immediate and quantitative structural insights.

Enhanced SERS performance of gold nanoparticle assemblies on a cysteine-mutant Tobacco mosaic virus scaffold

The employment of biomolecular templates for the synthesizing nanohybrid constructs is expanding, driven by their prospective uses in biosensing and biomedical fields. Gold nanoparticles (AuNPs) and, in particular, their assemblies are especially preferred for Surface Enhanced Raman Spectroscopy (SERS) because of their ability to amplify Raman signals through localized surface plasmon resonances, thus enabling the detection of molecules at exceedingly low concentrations. Our investigative approach is dedicated to studing the role of cysteine mutants in the nucleation and assembly of AuNPs on Tobacco mosaic virus (TMV-C, carrying T158C mutation) scaffolds. Employing biomineralization and direct grafting methods, we synthesized these nanohybrids and examined them using conventional transmission electron microscopy (TEM), in situ liquid TEM, and fluorescence spectroscopy. We demonstrated that the syntheses obtained with TMV-C give denser plasmonic nanostructures, with is ideal for SERS applications. The SERS performances of these novel nanohybrids with various AuNPs sizes and densities were evaluated, revealing excellent enhancement factors for the nanosystems obtained by direct grafting that highlight their potential for the detection of biomolecules in solution.

Role of the aging procedure on the ω + β microstructure in the metastable β Ti-30Nb-3Fe alloy

This report investigated the effect of low-temperature aging on the ω + β microstructure in the Ti-30Nb-3Fe alloy (wt%). Samples were subjected to several aging procedures. The ω + β microstructure remained quite stable under conventional transmission electron microscopy across most temperature/time regimes. Only the double aging treatment led to the growth of ω particles and an increased in Vickers hardness, indicating that the pre-aging treatment was necessary to promote rapid ω growth in the subsequent treatment. Furthermore, the early stages of solute partitioning that accompanies ω growth did not drastically increase hardness, suggesting that no embrittlement occurred.

(Invited) Nano-Characterization of the Internal Morphology of Polymer Electrolyte Membranes with Cryogenic Scanning Transmission Electron Microscopy

Polymer membranes play important roles in electrochemical devices such as polymer electrolyte fuel cells. In recent years, there has been a growing interest in alkaline anion exchange membrane (AAEM) fuel cells as the alkaline environment offers a potential low-cost alternative to commercial proton exchange membrane (PEM) fuel cells [1]. Nonetheless, optimization of polymer properties such as ion conductivity and mechanical durability is still necessary to achieve commercially viable AAEM based fuel cells. Often, semicrystalline polymer backbones are selected for AAEM design, aiming to replicate the exceptional performance of Nafion in PEM fuel cells where the crystalline domains are believed to act as cross-linkers, mitigating excessive water uptake and mechanical degradation [2,3].Characterizing the local polymer morphology at the nanoscale is a crucial step in understanding how to optimize the design of AAEMs with enhanced durability and ion conductivity. Imaging polymers using conventional transmission electron microscopy (TEM) techniques is challenging however, due to their inherently-high beam sensitivity and low image contrast. Here, we demonstrate nanometer-resolution mapping of the ordering in polymer electrolyte membranes using cryogenic four-dimensional scanning transmission electron microscopy (cryo-4D-STEM). Conventionally used for mapping inorganic crystalline materials, the 4D-STEM technique has more recently also found applications in mapping semicrystalline, soft materials as well [4,5]. Here, using a model system of semicrystalline hydrogenated polynorbornene (hPN) AAEMs [6], we demonstrate the effects of polymer synthesis including molecular weight and thermal history on crystalline morphology and in turn on the polymer performance. Further, we compare the crystalline architecture of the hPN copolymers to that of Nafion 212 (Figure 1). Our results suggest that polymers with homogeneously distributed, nanometer-size crystalline domains lead to improved water management, a property directly related to the mechanical integrity of the polymer, with possible impacts on ion conductivity. More broadly, the results give new insights into how we can tune AAEM design in order to improve their performance.[1] Dekel, Journal of Power Sources, 375, 158-169. (2018).[2] Yang, et al. Chemical Reviews, 122(6), 6117-6321. (2022).[3] Mauritz & Moore, Chemical reviews, 104(10), 4535-4586. (2004).[4] Panova, et al. Nature materials, 18(8), 860-865. (2019).[5] Bustillo, et al. Accounts of chemical research, 54(11), 2543-2551. (2021).[6] Treichel, et al. Macromolecules, 53(19), 8509-8518. (2020).Figure 1: Cryo-4D-STEM crystallinity maps of hPN-co-iPrMe copolymers with varying syntheses and Nafion 212. We observe improvement in the water management of the copolymer membranes as the crystalline domains are reduced in volume and distributed homogeneously. The chemical structures of the polymers are listed below the crystallinity maps. Figure 1

Facile in-Situ Straining Platform for Transmission Electron Microscopy with Quantification of Strains

This work aims to design and model an experimental platform to perform in-situ straining experiments in a transmission electron microscope (TEM), enabling structural characterization of materials under tensile strain. The ability to visualize changes in the crystal structure of materials under strain can provide important insights into failure modes and improve understanding of process-structure-property relationships. Existing methods of in-situ straining typical involve complex sample preparation and are not well-suited to the study of thin films or sensitive battery materials. In this work, thin films of materials are deposited on a conventional TEM grid adhered to a copper template. The template is mounted in a Gatan 654 single tilt straining holder and strained within the TEM. Electron diffraction patterns are taken from the specimens, and strain can be calculated based on shifts in the Bragg reflections.An essential component of in-situ straining experiments is the quantification of applied strains. For our facile straining platform, we calculate the expected values of strain on our specimen using a finite element model. The finite element strain simulation data was compared with experimental data collected during in-situ straining experiments. Experimental results on a variety of material systems, including those applicable to lithium-ion batteries, are within the bounds of our model. Overall, the ability to accurately simulate in-situ strains using the finite element model enables researchers to better explain observation in the TEM, improve the reproducibility of the experiments, and optimize straining geometries to be tested. Figure 1

Parallel-beams magnetic actuator for in-situ transmission electron microscope observation of mechanical testing

Abstract Understanding the microscopic mechanisms behind mechanical fractures is essential for enhancing material properties and increasing reliability through fatigue suppression. Conventional mechanical testing methods, such as indentation tests that press a sharp needle into a specimen or tensile tests using hydraulic pumps, are unable to capture nanoscale deformations under applied forces. As a result, the microscopic mechanisms that influence mechanical properties are often inferred indirectly, and material design largely depends on the engineer’s intuition and occasional serendipity. To overcome these challenges, in-situ observation techniques utilizing transmission electron microscopes (TEMs) have been developed to enable the observation of sample deformations at the nanoscale. However, despite their high resolution, conventional TEMs are limited by a small available space -often just a few millimeters- that restricts the application of sufficient force to fracture specimens. Traditional actuation methods, such as thermal expansion, electrostatic force, and piezoelectric actuators, fail to generate significant forces within such confined spaces. In response to these limitations, our research involved the development of a micromachine with multiple parallel beams. This device leverages the Laplace force generated by an electric current passing through the beams and the magnetic field of the TEM. We demonstrated the capability to produce significant force using the magnetic field from the microscope’s magnetic lens. The actuator developed in our study successfully generated forces exceeding 50 µN, marking a significant advancement in the in-situ observation capabilities for mechanical testing.

Wehnelt photoemission in an ultrafast electron microscope: Stability and usability

We tested and compared the stability and usability of three different cathode materials and configurations in a thermionic-based ultrafast electron microscope: (1) on-axis thermionic and photoemission from a custom 100 μm diameter LaB6 source with a graphite guard ring, (2) off-axis photoemission from the Ni aperture surface of the Wehnelt electrode, and (3) on-axis thermionic and photoemission from a custom 200 μm diameter polycrystalline Ta source. For each cathode type and configuration, including the Ni Wehnelt aperture, we illustrate how the photoelectron beam-current stability is deleteriously impacted by simultaneous cooling of the source following thermionic heating. Furthermore, we demonstrate usability via collection of parallel- and convergent-beam electron diffraction patterns and by formation of the optimum probe size. We find that usability of the off-axis Ni Wehnelt-aperture photoemission is at least comparable to on-axis LaB6 thermionic emission, as well as to on-axis photoemission [the heretofore conventional approach to ultrafast electron microscopy (UEM) in thermionic-based instruments]. However, the stability and achievable beam currents for off-axis photoemission from the Wehnelt aperture were superior to that of the other cathode types and configurations, regardless of the electron-emission mechanism. Beam-current stability for this configuration was found to be ±1% (one standard deviation from the mean) for 70 min (longest duration tested), and steady-state beam current was reached within the sampling-time resolution used here (∼1 s) for 15 pA beam currents (i.e., 460 electrons per packet for a 200 kHz repetition rate). Repeatability and robustness of the steady-state condition were also found to be within ±1% of the mean. We discuss the implications of these findings for UEM imaging and diffraction experiments, for pulsed-beam damage measurements, and for practical switching between optimum conventional TEM and UEM operation within the same instrument.

Moiré fringe imaging of heterostructures by scanning transmission electron microscopy

A heterostructured crystalline bilayer specimen is known to produce moiré fringes (MFs) in the conventional transmission electron microscopy (TEM). However, the understanding of how these patterns form in scanning transmission electron microscopy (STEM) remains limited. Here, we extended the double-scattering model to establish the imaging theory of MFs in STEM for a bilayer sample and applied this theory to successfully explain both experimental and simulated STEM images of a perovskite PbZrO3/SrTiO3 system. Our findings demonstrated that the wave vectors of electrons exiting from Layer-1 and their relative positions with the atomic columns of Layer-2 should be taken into account. The atomic column misalignment leads to a faster reduction in the intensity of the secondary scattering beam compared to the single scattering beam as the scattering angle increases. Consequently, the intensity distribution of MFs in the bright field (BF)-STEM can be still described as the product of two single atomic images. However, in high angle annular dark field (HAADF)-STEM, it is approximately described as the superposition of the two images. Our work not only fills a knowledge gap of MFs in incoherent imaging, but also emphasizes the importance of the coherent scattering restricted by the real space when analyzing the HAADF-STEM imaging.

Investigation of the structural stability of polycrystalline cBN under near-industrial grinding process conditions

Cubic boron nitride (cBN) is an important material in the cutting and metal processing industries due to its exceptional hardness and high thermal stability, which allows it to withstand temperatures up to 1400 °C without decomposing. These properties make cBN and, with that, polycrystalline cBN (PcBN) ideal materials for high-speed machining and other applications, resulting in increased efficiency and reduced costs in production processes. Despite its established importance, the possibility of phase transformations into hexagonal boron nitride phases or partial amorphization during industrial PcBN grinding processes remains unclear and, due to the deteriorated mechanical characteristics, raised recently some concerns about possible losses of cBN's hardness, wear resistance, and abrasive properties. In order to address this issue and for identifying such potential structural changes, a commercial PcBN grade was exposed to near-industrial grinding process conditions and then characterized in detail by conventional (scanning) transmission electron microscopy (S)TEM and (scanning) electron diffraction techniques. To understand the influence of the PcBN machining on the material, the grinding process also needs to be assessed via the identification of chip formation mechanisms, which is as well addressed in this work. The main outcome of this study, combining a material analysis and a manufacturing technology point of view, is that there is so far no evidence of any structural instabilities of PcBN under usual processing conditions.

Comparison of differential phase Contrast, Lorentz and non-Lorentz transmission electron microscopy for microstructural and magnetic imaging of alnico magnet

In this work, non-Lorentz, Lorentz transmission electron microscopy (LTEM) and differential phase contrast (DPC) methods were used to observe the phase and magnetic structure of the alnico magnet. All observations were performed on alnico-5 in transverse orientation. The results showed the magnetic contrast of the domain walls in the conventional TEM images despite the effects of the lens magnetic field, which was verified and explained using DPC. Experimental methodology and sample results are summarized with advice for further research.

Particle morphology characterization in an over-aged Al-Cu-Mg-Si alloy using TEM

A quantitative analysis of particle morphologies in a heterophase Al-Cu-Mg-Si alloy after over-aging has been performed using conventional transmission electron microscopy (TEM) imaging in dark- and bright-field modes. An appropriate analytical procedure has been suggested separating {100}Al plate- and 〈100〉Al rod-shaped particles using TEM images taken along 〈100〉Al and 〈110〉Al zone axes. The obtained statistical particle morphology parameters were verified by macroscopic strength contribution calculations.

Reducing two-level systems dissipations in 3D superconducting niobium resonators by atomic layer deposition and high temperature heat treatment

Superconducting qubits have arisen as a leading technology platform for quantum computing, which is on the verge of revolutionizing the world's calculation capacities. Nonetheless, the fabrication of computationally reliable qubit circuits requires increasing the quantum coherence lifetimes, which are predominantly limited by the dissipations of two-level system defects present in the thin superconducting film and the adjacent dielectric regions. In this paper, we demonstrate the reduction of two-level system losses in three-dimensional superconducting radio frequency niobium resonators by atomic layer deposition of a 10 nm aluminum oxide Al2O3 thin films, followed by a high vacuum heat treatment at 650 °C for few hours. By probing the effect of several heat treatments on Al2O3-coated niobium samples by x-ray photoelectron spectroscopy plus scanning and conventional high resolution transmission electron microscopy coupled with electron energy loss spectroscopy and energy dispersive spectroscopy, we witness a dissolution of niobium native oxides and the modification of the Al2O3-Nb interface, which correlates with the enhancement of the quality factor at low fields of two 1.3 GHz niobium cavities coated with 10 nm of Al2O3.

Towards Construction of a Novel Nanometer-Resolution MeV-STEM for Imaging Thick Frozen Biological Samples

Driven by life-science applications, a mega-electron-volt Scanning Transmission Electron Microscope (MeV-STEM) has been proposed here to image thick frozen biological samples as a conventional Transmission Electron Microscope (TEM) may not be suitable to image samples thicker than 300–500 nm and various volume electron microscopy (EM) techniques either suffering from low resolution, or low speed. The high penetration of inelastic scattering signals of MeV electrons could make the MeV-STEM an appropriate microscope for biological samples as thick as 10 μm or more with a nanoscale resolution, considering the effect of electron energy, beam broadening, and low-dose limit on resolution. The best resolution is inversely related to the sample thickness and changes from 6 nm to 24 nm when the sample thickness increases from 1 μm to 10 μm. To achieve such a resolution in STEM, the imaging electrons must be focused on the specimen with a nm size and an mrad semi-convergence angle. This requires an electron beam emittance of a few picometers, which is ~1000 times smaller than the presently achieved nm emittance, in conjunction with less than 10−4 energy spread and 1 nA current. We numerically simulated two different approaches that are potentially applicable to build a compact MeV-STEM instrument: (1) DC (Direct Current) gun, aperture, superconducting radio-frequency (SRF) cavities, and STEM column; (2) SRF gun, aperture, SRF cavities, and STEM column. Beam dynamic simulations show promising results, which meet the needs of an MeV-STEM, a few-picometer emittance, less than 10−4 energy spread, and 0.1–1 nA current from both options. Also, we designed a compact STEM column based on permanent quadrupole quintuplet, not only to demagnify the beam size from 1 μm at the source point to 2 nm at the specimen but also to provide the freedom of changing the magnifications at the specimen and a scanning system to raster the electron beam across the sample with a step size of 2 nm and the repetition rate of 1 MHz. This makes it possible to build a compact MeV-STEM and use it to study thick, large-volume samples in cell biology.

Eosinophil activation during immune responses: an ultrastructural view with an emphasis on viral diseases.

Eosinophils are cells of the innate immune system that orchestrate complex inflammatory responses. The study of the cell biology of eosinophils, particularly associated with cell activation, is of great interest to understand their immune responses. From a morphological perspective, activated eosinophils show ultrastructural signatures that have provided critical insights into the comprehension of their functional capabilities. Application of conventional transmission electron microscopy in combination with quantitative assessments (quantitative transmission electron microscopy), molecular imaging (immunoEM), and 3-dimensional electron tomography have generated important insights into mechanisms of eosinophil activation. This review explores a multitude of ultrastructural events taking place in eosinophils activated in vitro and in vivo as key players in allergic and inflammatory diseases, with an emphasis on viral infections. Recent progress in our understanding of biological processes underlying eosinophil activation, including in vivo mitochondrial remodeling, is discussed, and it can bring new thinking to the field.

Electron-translucency and partial defects of synaptic basal lamina in the electrocyte synapse of an electric ray (Narke japonica) in 3D embedment-free section electron microscopy.

The synaptic basal lamina of the electrocytes was disclosed to be electron-translucent to some extent when viewed in an en-face direction in embedment-free section transmission electron microscopy (EFS-TEM), and synaptic vesicles located close to the presynaptic membrane were seen through the synaptic basal lamina together with the presynaptic and postsynaptic membranes. This feature of translucency has the potential to analyze possible spatial interrelations in situ between bioactive molecules in the synaptic basal lamina and the synaptic vesicles in further studies. The synaptic basal lamina, appearing as an electron-dense line sandwiched by two parallel lines representing the presynaptic and postsynaptic membranes in ultrathin sections cut right to the synaptic junctional plane in conventional TEM, was not fully continuous but randomly intermittent along its trajectory. Compatible with the intermittent line appearance, the en-face 3D view in embedment-free section TEM revealed for the first time partial irregular defects of the synaptic basal lamina. Considering the known functional significance of several molecules contained in the synaptic basal lamina in the maintenance and exertion of the synapse, its partial defects may not represent its rigid structural features, but its immature structure under remodeling or its dynamic changes in consistency such as the sol/gel transition, whose validity needs further examination. RESEARCH HIGHLIGHTS: In embedment-free section TEM, a 3D en-face view of synaptic basal lamina in situ is reliably possible. The basal lamina en-face is electron-translucent, which makes it possible to analyze spatial interrelation between pre- and post-synaptic components. Partial irregular defects in the basal lamina are revealed in Torpedo electrocytes, suggesting its remodeling or dynamic changes in consistency.

The proteome of bacterial membrane vesicles in Escherichia coli-a time course comparison study in two different media.

Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment. Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions. Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

Deep learning-enabled probing of irradiation-induced defects in time-series micrographs

Modeling time-series data with convolutional neural networks (CNNs) requires building a model to learn in batches as opposed to training sequentially. Coupling CNNs with in situ or operando techniques opens the possibility of accurately segmenting dynamic reactions and mass transport phenomena to understand how materials behave under the conditions in which they are used. In this article, in situ ion irradiation transmission electron microscopy (TEM) images are used as inputs into the CNN to assess the defect generation rate, defect cluster density, and saturation of defects. We then use the output segmentation maps to correlate with conventional TEM micrographs to assess the model’s ability to detail nanoscale interactions. Next, we discuss the implications of preprocessing and hyperparameters on model variability, accuracy when expanded to other datasets, and the role of regularization when controlling model variance. Ultimately, we eliminate human bias when extrapolating physical metrics, speed up analysis time, decouple reactions that happen at 100 ms intervals, and deploy models that are both accurate and transferable to similar experiments.