Standard Transmission Electron Microscopy Research Articles

SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients’ surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Innovative stain-free technique for high-resolution imaging of virus particles via standard transmission electron microscopy

This research presents a groundbreaking approach in virus-related research, addressing challenges in electron microscopy (EM). This imaging technique has been crucial in exploring virus structures; however, traditional methods involve complex sample preparations and the risk of contamination. Herein, we introduce an approach that overcomes these obstacles, enabling high-resolution virus imaging without toxic staining procedures. Focusing on Begomovirus particles, an economically significant plant virus genus, our images confirm their non-enveloped structure and their twin icosahedral symmetry. Our methods involve sample collection, purification, and crystallization, followed by transmission electron microscopy - selected area electron diffraction (TEM-SAED) analysis. Notably, this study achieves 2D and 3D virus imaging through standard TEM, providing a new avenue for virus structure analysis and advancing virus-related research. Remarkable high image quality stemmed from the crystallization process, offering exciting possibilities for improving virus research and diagnosis while eliminating staining limitations.

Carbon nanoparticle identification using transmission electron microscopy methods in biological samples

Carbon nanoparticles are a common type of nanoparticles, the identification of which in biological samples is associated with great difficulties. It is demonstrated that the use of standard transmission electron microscopy in combination with the electron diffraction method is a reliable and relevant tool for the carbon nanoparticles identification in biological samples.

Ultrastructure of receptive stigma and transmitting tissue at anthesis in two pear species

The ultrastructure of stigmatic and stylar secretory tissues was studied in one cultivar of <i>Pyrus communis</i> and six cultivars of <i>Pyrus pyrifolia</i> var. <i>culta</i> (the so-called Nashi pear) using standard light, scanning, and transmission electron microscopy methods. Although both tissues secreted an extracellular fluid necessary for the development of male gametophyte, they differed markedly in the ultrastructure of the extracellular matrix and in the distribution and ultrastructure of organelles. The difference was most evident in regard to the endoplasmic reticulum, which represented the rough, smooth, and vesicular type in stigmatic papillate epidermis and distal stigmatoid tissue cells and occurred mainly as the rough type in form of expanded cisternae filled with fine-fibrillar content in the transmitting tissue of the style.

Wide-gamut structural colours on oakblue butterflies by naturally tuned photonic nanoarchitectures.

The iridescent structural colours of butterflies, generated by photonic nanoarchitectures, often function as species-specific sexual signals; therefore, they are reproduced precisely from generation to generation. The wing scales of oakblue hairstreak butterflies (genus Arhopala, Theclinae, Lycaenidae, Lepidoptera) contain multi-layer photonic nanoarchitectures, which can generate a wide range of structural colours, from violet to green. By scanning (SEM) and cross-sectional transmission electron microscopy (TEM) investigation, the colour tuning mechanism of the cover scales was explored. We revealed that the characteristic size change of structural elements in similar photonic nanoarchitectures led to different structural colours that were examined by various reflectance spectrophotometry techniques. The measured structural properties of the naturally tuned photonic nanoarchitectures were used to calculate wing reflectances, which were compared with the measurement results. We found that the simulated structural colours were systematically redshifted by 95-126 nm as compared with the measured normal-incidence reflectance results. This is attributed to the swelling of the chitinous multi-layer structures during the standard TEM sample preparation and the tilt of the cover scales, which both affect the apparent layer thicknesses in the TEM cross-sections. We proposed a simulation correction and compared the results with the layer thicknesses measured on cryogenically prepared non-embedded SEM cross-sections.

Landmark-based retrieval of inflamed skin vessels enabled by 3D correlative intravital light and volume electron microscopy

The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Several methods have been developed to overcome these limitations, mainly by correlating the light microscopical image to the electron microscope with correlative light and electron microscopy (CLEM) techniques. Since there is more than one method to obtain the region of interest (ROI), the workflow must be adjusted according to the research question and biological material addressed. Here, we describe in detail the development of a three-dimensional CLEM workflow for mouse skin tissue exposed to an inflammation stimulus and imaged by intravital microscopy (IVM) before fixation. Our aim is to relocate a distinct vessel in the electron microscope, addressing a complex biological question: how do cells interact with each other and the surrounding environment at the ultrastructural level? Retracing the area over several preparation steps did not involve any specific automated instruments but was entirely led by anatomical and artificially introduced landmarks, including blood vessel architecture and carbon-coated grids. Successful retrieval of the ROI by electron microscopy depended on particularly high precision during sample manipulation and extensive documentation. Further modification of the TEM sample preparation protocol for mouse skin tissue even rendered the specimen suitable for serial block-face scanning electron microscopy (SBF-SEM).

Ultrastructure of Egg Envelopes and Early Embryos of Rohdella amazonica (Trematoda: Aspidogastrea) Parasitic in Banded Puffer Fish, Colomesus psittacus

Egg structure and early embryonic development of the aspidogastrean, Rohdella amazonica, a basal trematode, were studied by transmission electron microscopy (TEM) to gain insight into functional, developmental, and phylogenetic characteristics. Gravid worms were removed from the intestine of naturally infected banded puffer fish Colomesus psittacus, collected from the Bay of Marajó, Paracauari River (Pará, Brazil) and processed by standard TEM methods. By the time of pronuclear fusion, the fertilized zygote was already enclosed in a thick, electron-dense pre-operculate eggshell and an underlying layer of vitellocytes that fused into a vitelline syncytium as they were still secreting their shell granules. When cleavage commenced, a small number of macromeres moved to the area just underneath the eggshell, where they fused to form a single syncytial embryonic envelope. Simultaneously, the smaller blastomeres continued to divide as they maintained contact with each other, but remained separate from the vitelline syncytium. Concurrent with these cellular changes, a thickened knob expanded at one pole of the eggshell and began to form an opercular suture. By the time the operculum was fully formed, the vitelline syncytium had mostly degenerated, while the smaller blastomeres had become cohesive as a single mass that preceded the differentiation and morphogenesis of the cotylocidium larva. The general pattern of cleavage and eggshell formation resembles that of other trematodes and polylecithal cestodes, but the single embryonic envelope has been reported only in a few basal taxa. The only other aspidogastrean studied in detail to date is very similar, indicating close phylogenetic affinity and conservatism within this basal neodermatan and neoophoran group.

Analysis of the extranucleolar ribonucleoprotein particles of Cycas revoluta Thunb. (Cycadaceae) and Ceratozamia mexicana Brongn. (Zamiaceae)

Background: Nuclear ribonucleoprotein particles play a key role in RNA processing and in the gene expression pathway. Interchromatin granules (GICs) involved in the metabolism of pre-messenger RNA (pre-mRNA) were described in Allium cepa and Chiranthodendron pentadactylon . Other particles as Lacandonia granules (LGs) were found in Lacandonia schismatica as well as Ginkgo biloba and Welwitschia mirabilis . LGs are structures equivalent to perichromatin granules (PCGs) described in mammals and to Balbiani ring granules (BRGs) described in the midge Chironomus tentans . PCGs and BRGs are involved in the metabolism of messenger RNA (mRNA). Here, we analyze the extranucleolar particles from Cycas revoluta and Ceratozamia mexicana and compare them to GICs and LGs using conventional electron microscopy and atomic force microscopy.
 Species study: Cycas revoluta (Cycadaceae) and Ceratozamia mexicana (Zamiaceae)
 Hypothesis: The extranucleolar ribonucleoprotein particles in the nuclei of C. revoluta and C. mexicana are equivalent to GICs or GLs.
 Methods: Fragments of young leaves of C. revoluta and C. mexicana were processed for standard transmission electron microscopy. Thin sections were stained with the EDTA technique preferential for ribonucleoproteins and osmium amine specific for DNA. From the semithin sections the samples were studied with the AFM and images of them were obtained.
 Results: Ribonucleoprotein particles 32 nm in diameter are present in the interchromatin and perichromatin space in C. revoluta and C. mexicana .
 Conclusion: Ribonucleoprotein particles present in the cell nuclei of C. mexicana and C. revoluta are ultrastructurally equivalent to LGs .
 
 
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Designing Sb<sub>2</sub>Te<sub>3</sub> heterophase homostructure

<p indent="0mm">Antimony sesquitelluride (Sb₂Te₃) is the most important hosting material for the development of high-performance phase-change memory and brain-like computing technology. This material has two crystalline phases, namely, the metastable cubic rock-salt structure with 1/6 atomic sites taken by randomly distributed vacancies, and the stable rhombohedral phase with layered structures and vacant gaps in hexagonal stacking. The former is an Anderson insulator (AI) and serves as the key ingredient for memory applications, while the latter is a prototypical topological insulator (TI). In this work, we propose a new heterophase homostructure concept for Sb₂Te₃, which in principle could utilize both the topologically protected surface state of the rhombohedral phase and the Anderson localization features of the cubic phase in the bulk interior to achieve ultralow-loss electron transport. To sustain the topological surface state, the edges of the device should be kept in rhombohedral phase with the thickness larger than <sc>~4 nm.</sc> The bulk interior of the sample should be transformed into cubic phase with strong electron localization. According to our density functional theory (DFT) results, the layered atomic building blocks for the two phases are quite similar, though their stacking sequence and degree of structural disorder are different. Thanks to the similar structure and mass density of the two phases, lattice mismatch is not expected to be present at the interface of the heterophase homostructure, superior to heterostructures made of different materials. To lend support to this heterophase homostructure concept, we have prepared a <sc>~500 nm</sc> thick rhombohedral Sb₂Te₃ thin film on a silicon substrate via molecular beam epitaxy (MBE) and made an attempt to induce the phase transition in its bulk interior. A cross-sectional specimen for transmission electron microscopy (TEM) experiments was prepared by a dual beam focused ion beam (FIB) system. The TEM specimen along the electron beam direction was <sc>~80 nm.</sc> For standard TEM observation in short time, the damage brought by electron beam is very limited. However, when the electron beam was focused in an exposure area of <sc>~120 nm</sc> or below inside an electron microscope with a fixed accelerating voltage of <sc>200 keV,</sc> visible structural changes in the Sb₂Te₃ specimen were observed. A gradual structural transition from the stable rhombohedral phase to the metastable cubic phase was observed under extensive electron beam irradiation over <sc>90 min.</sc> The<italic> in situ </italic>high-resolution TEM experiments revealed a clear vacancy disordering induced metastabilization process in the Sb₂Te₃ specimen, where Sb atoms from the atomic stacks diffused into the vacant gap layers in Sb₂Te₃ and eventually triggered a structural change from rhombohedral to cubic Sb₂Te₃. Our DFT calculations corroborated this picture, which showed a systematic increase in total energy as the vacant gap was gradually filled in rhombohedral Sb₂Te₃. The bonding environment of the Sb atoms in the gap for the two phases is entirely different. For the cubic phase, the Sb atoms in the gap are located as the center of octahedral structures with the bond angle of ~90°, which is consistent with the Sb atoms in the original rhombohedral and cubic phase. In stark contrast, the Sb atoms in the gap layers of rhombohedral phase are in a distorted bonding environment with bond angles of both ~110° and ~90°, leading to a large energy penalty for the rhombohedral phase. When the concentration of vacancies in the target layers reached 50%–70%, a change in stacking sequence from rhombohedral to cubic stacking was triggered. The critical vacancy concentration was mainly affected by the degree of compressive strain perpendicular to the target layers. In summary, we have discussed the design concept of a Sb₂Te₃ based heterophase homostructure and demonstrated that this structure could be free of lattice mismatch via <italic>in situ</italic> TEM experiments and DFT calculations. Finally, we provided some guidelines for practical synthesis of Sb₂Te₃ heterophase homostructure samples to explore the interaction between topological physics and Anderson localization of electrons, and to fabricate potential electronic devices of ultra-low power consumption.

The ultrastructural development and 3D reconstruction of the transparent carapace of the ostracod Skogsbergia lerneri

The Skogsbergia lerneri is a marine ostracod which possesses a carapace that is both protective and transparent. Since development of this carapace and how it is maintained in the adult is not known, the aim of this investigation was to carry out an in-depth ultrastructural study of the ostracod carapace at different developmental stages. Standard transmission electron microscopy and novel serial block face scanning electron microscopy (SBF-SEM) were undertaken to discern carapace ultrastructure in both two and three dimensions. Analysis revealed a carapace consisting of the same basic layer structure as other myodocopid ostracods, namely an epicuticle, exocuticle, endocuticle and membranous layer, but with a thinner adult carapace of mean thickness of 19.2 ± 1.78 µm, n = 5. The carapace layers, except for instar 1 ostracods, had similar relative proportions throughout development. The endocuticle and membranous layer thickened through advancing developmental stages due to an increase in calcified crystalline polyhedrons and a greater number of chitinous lamellae in the membranous layer. Crystalline polyhedron dimensions were significantly smaller near the boundary with the membranous layer. The borders between the carapace layers were indistinct; SBF-SEM revealed an abundance of epicuticle projections into the exocuticle and apparent gradual merging at the boundary of the exocuticle and the endocuticle. Here, we discuss how the S. lerneri carapace layer structure has evolved to serve a specific mechanical function, allowing surface protection and rigidity. In addition, we suggest that the lack of pigment and graduated layer boundaries contribute to the transparency of the carapace.

Focused Ion Beam-Scanning Electron Microscopy for Investigating Plasmodesmal Densities.

Plasmodesmata (PD) facilitate the exchange of nutrients and signaling molecules between neighboring plant cells, and they are therefore essential for proper growth and development. PD have been studied extensively in efforts to elucidate the ultrastructure of individual PD nanopores and the distribution of PD in a variety of cell walls. These studies often involved the use of serial ultrathin sections and manual quantification of PD by transmission electron microscopy (TEM). In recent years, a variety of techniques that offer more amenable approaches for quantifying PD distribution have been reported. Here, we describe the quantification of PD densities using the serial scanning electron microscopy technique called focused ion beam-scanning electron microscopy (FIB-SEM). For this, resin-embedded samples prepared by standard TEM methods undergo successive rounds of imaging by SEM interspersed with milling of the sample surface by a focused beam of gallium ions to reveal a new surface. In this way, the details of the sample are sequentially revealed and imaged. Over the course of a few hours, repetitive milling and imaging facilitates the automated collection of nanometer-resolution data of several μm of sample depth. FIB-SEM can be targeted to interrogate specific cell walls and cell wall junctions, and the subsequent three-dimensional renderings of the data can be used to visualize the ultrastructural details of the sample. PD densities can then be rapidly quantified by calculating the number of PD per μm2 of cell wall observed in the renderings.

Morphological Characteristics of Idiopathic Inflammatory Myopathies in Juvenile Patients.

Background: In juvenile idiopathic inflammatory myopathies (IIMs), morphological characteristic features of distinct subgroups are not well defined. New treatment strategies require a precise diagnosis of the subgroups in IIM, and, therefore, knowledge about the pathomorphology of juvenile IIMs is warranted. Methods: Muscle biopsies from 15 patients (median age 8 (range 3–17) years, 73% female) with IIM and seven controls were analyzed by standard methods, immunohistochemistry, and transmission electron microscopy (TEM). Detailed clinical and laboratory data were accessed retrospectively. Results: Proximal muscle weakness and skin symptoms were the main clinical symptoms. Dermatomyositis (DM) was diagnosed in 9/15, antisynthetase syndrome (ASyS) in 4/15, and overlap myositis (OM) in 2/15. Analysis of skeletal muscle tissues showed inflammatory cells and diffuse upregulation of MHC class I in all subtypes. Morphological key findings were COX-deficient fibers as a striking pathology in DM and perimysial alkaline phosphatase positivity in anti-Jo-1-ASyS. Vascular staining of the type 1 IFN-surrogate marker, MxA, correlated with endothelial tubuloreticular inclusions in both groups. None of these specific morphological findings were present in anti-PL7-ASyS or OM patients. Conclusions: Morphological characteristics discriminate IIM subtypes in juvenile patients, emphasizing differences in aetiopathogenesis and supporting the notion of individual and targeted therapeutic strategies.

100 keV vacuum sealed field emission gun for high resolution electron microscopy

A standalone 100 kV field emission gun (FEG) has been developed that can be installed and operated on a standard transmission electron microscopy electron optical column or custom designed high voltage electron optical columns. The FEG comprises a thermally assisted field emission cathode and an asymmetric electrostatic lens that can operate from 20 to 100 kV in an ultrahigh vacuum (UHV) chamber. In its current configuration, the FEG has spherical and chromatic aberration coefficients (Cs and Cc, respectively) in the range of Cs = 607–670 mm and Cc = 60–87 mm at 100 keV over a range of working distances of 50–206 mm from the exit plane of the FEG unit. A dedicated high voltage supply unit with voltage ripples of less than 1 ppm at 100 kV has also been developed. The FEG is transported under UHV and does not require the use of SF6 gas during operation, as is customary in high voltage FEG TEMs. Preliminary results of operating the FEG on a Philips Tecnai 12 and a JEOL JEM-1400HR TEM show the resolution of gold (111) crystal planes at 0.235 nm and (200) planes at 0.202 nm.

Review of Post-embedding Immunogold Methods for the Study of Neuronal Structures.

The post-embedding immunogold (PI) technique for immunolabeling of neuronal tissues utilizing standard thin-section transmission electron microscopy (TEM) continues to be a prime method for understanding the functional localization of key proteins in neuronal function. Its main advantages over other immunolabeling methods for thin-section TEM are (1) fairly accurate and quantifiable localization of proteins in cells; (2) double-labeling of sections using two gold particle sizes; and (3) the ability to perform multiple labeling for different proteins by using adjacent sections. Here we first review in detail a common method for PI of neuronal tissues. This method has two major parts. First, we describe the freeze-substitution embedding method: cryoprotected tissue is frozen in liquid propane via plunge-freezing, and is placed in a freeze-substitution instrument in which the tissue is embedded in Lowicryl at low temperatures. We highlight important aspects of freeze-substitution embedding. Then we outline how thin sections of embedded tissue on grids are labeled with a primary antibody and a secondary gold particle-conjugated antibody, and the particular problems encountered in TEM of PI-labeled sections. In the Discussion, we compare our method both to earlier PI methods and to more recent PI methods used by other laboratories. We also compare TEM immunolabeling using PI vs. various pre-embedding immunolabeling methods, especially relating to neuronal tissue.

Spermatozoon of the wild scalloped perchlet, Ambassis nalua (Hamilton, 1822): Ultrastructure and morphometric analysis

The description of sperm morphology is fundamental in the reproductive biology of fishes, but this information is limited in the family Ambassidae. Our report hence focused on the ultrastructure and morphometric analysis of spermatozoa in a pelagic fish Ambassis nalua. All fish (n = 75) were obtained during January and March 2017 from the Estuarine Pranburi River, Thailand. The standard length of fish used in this study was 3.4 ± 0.12 cm (mean ± standard deviation). All specimens were considered mature based on the abundance of spermatozoa in the testis. The testicular organs were collected and observed using standard histology and transmission electron microscopy (TEM). Ultrastructural observation associated with morphometric analysis showed that spermatozoa are structurally long cells of approximately 51.17 ± 4.54 µm total length, composed of a head, a midpiece and a tail. The head had no acrosome, and the granular structure of condensed chromatin was observed within the ovoid nucleus. The midpiece consisted of a short cylindrical region with the length of 1.29 ± 0.87 µm in diameter, having the centriolar complex organization and eight mitochondria (approx. 0.32 ± 0.02 µm each). The uniflagellar tail was clearly identified with a classical 9+2 arrangements of microtubules. Based on these characteristics, the spermatozoon of wild scalloped perchlet are considered as uniflagellate anacrosomal aquqsperm. The morphological features, including the number of mitochondria, may be used for further cryopreservation and in the evolutionary biology of this species.

Magnetic imaging of various topological spin textures and their dynamics

Nanometer-scale vortex-like topological spin textures, such as magnetic skyrmion and antiskyrmion, have recently attracted enormous attention owing to their topological nature and emergent electromagnetic properties. To confirm such minute twisted spin textures and their dynamics under external stimuli, real-space imaging techniques with high spatial resolution and high recording speed are required.To realize the Bloch-type magnetic skyrmion with a topological number of − 1 (where spins swirl from the north-pole in the core to the south-pole in the peripheral, and wrap a sphere), we have developed a technique allowing not only to create skyrmions via precise control of the magnetic field in a standard transmission electron microscope (TEM), but also to directly demonstrate their twisted textures and dynamics. We employed a phase imaging technique, called differential-phase contrast, in the scanning TEM mode to quantitatively characterize Néel-type magnetic twists, such as Bloch lines, that constitute the antiskyrmion with a topological number + 1. Furthermore, we took advantage of cryogenic high-resolution Lorentz TEM to probe atomic-scale skyrmions in a centrosymmetric magnet with a Ruderman–Kittel–Kasuya–Yosida coupling.To manipulate and track individual skyrmions and their lattice using a relatively low electric current, combined with field-quenching of skyrmions, we designed a thin FeGe microdevice having a notch, in which the spin current was localized in the specific area near a corner of the notch. Using this device, we tracked drift, Hall, and torque motions of single 80-nm-sized skyrmions and their bunches using electric current densities three orders of magnitude lower than those required to manipulate magnetic domain walls.

Golgi apparatus components in Entamoeba histolytica and Entamoeba dispar after monensin treatment.

Highly dynamic ribosomes, glycogen granules, thinly fibrillar material, and multiple membrane-bound vesicles are embedded in the matrix-rich cytoplasm of Entamoeba spp. trophozoites. The absence of a Golgi apparatus in these amoebae has been commonly accepted. Here we challenge this observation by incubating Entamoeba histolytica and Entamoeba dispar with monensin, an ionophore that produces swelling of the Golgi apparatus. We observe changes in the trophozoites through standard transmission electron microscopy, cryofixation and cryosubstitution, and analyze the label and expression of known resident proteins of the cis-GM130 and trans-TGN38 Golgi network through confocal microscopy and Western blot assays. Cryosubstitution and standard methods using the treatment, preserved membranous lamellae resembling Golgi components. GM130 and TGN38 Golgi antigens were found by immunoelectron, immunoblot, and co-localization by confocal microscopy using the reagent NBD C6-ceramide. Our results indicate that previously undetected Golgi apparatus components are present in the cytoplasm of E. histolytica and E. dispar.

Nd:YAG laser ablation of micro-crystalline graphite in a water suspension

The aim of this work was to obtain graphene-like phases (defected graphene, graphene oxide and reduced graphene oxide) as fine suspensions by applying pulsed laser ablation (PLA) of micro-crystalline graphite suspension in water medium. The fundamental (λ = 1064 nm), second (λ = 532 nm), third (λ = 355 nm) and fourth harmonics (λ = 266 nm) of a Nd:YAG laser system (15-ns pulse duration and 10-Hz pulse repetition rate) were used. The morphology of the particles was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Their phase composition and structure were explored by Raman and X-ray photoelectron spectroscopies, grazing incidence X-ray diffractometry (GIXRD) and TEM. The specimens were prepared by drop-casting on a glass substrate (for Raman and GIXRD studies) and on a standard TEM copper mesh for SEM and TEM examination.

Single-molecule imaging and kinetic analysis of intermolecular polyoxometalate reactions.

We induce and study reactions of polyoxometalate (POM) molecules, [PW12O40]3− (Keggin) and [P2W18O62]6− (Wells–Dawson), at the single-molecule level. Several identical carbon nanotubes aligned side by side within a bundle provided a platform for spatiotemporally resolved imaging of ca. 100 molecules encapsulated within the nanotubes by transmission electron microscopy (TEM). Due to the entrapment of POM molecules their proximity to one another is effectively controlled, limiting molecular motion in two dimensions but leaving the third dimension available for intermolecular reactions between pairs of neighbouring molecules. By coupling the information gained from high resolution structural and kinetics experiments via the variation of key imaging parameters in the TEM, we shed light on the reaction mechanism. The dissociation of W–O bonds, a key initial step of POM reactions, is revealed to be reversible by the kinetic analysis, followed by an irreversible bonding of POM molecules to their nearest neighbours, leading to a continuous tungsten oxide nanowire, which subsequently transforms into amorphous tungsten-rich clusters due to progressive loss of oxygen atoms. The overall intermolecular reaction can therefore be described as a step-wise reductive polycondensation of POM molecules, via an intermediate state of an oxide nanowire. Kinetic analysis enabled by controlled variation of the electron flux in TEM revealed the reaction to be highly flux-dependent, which leads to reaction rates too fast to follow under the standard TEM imaging conditions. Although this presents a challenge for traditional structural characterisation of POM molecules, we harness this effect by controlling the conditions around the molecules and tuning the imaging parameters in TEM, which combined with theoretical modelling and image simulation, can shed light on the atomistic mechanisms of the reactions of POMs. This approach, based on the direct space and real time chemical reaction analysis by TEM, adds a new method to the arsenal of single-molecule kinetics techniques.

Low-angle X-ray scattering for the determination of the size of mesoporous silica nanoparticles

In the last two decades, Mesoporous Silica Nanoparticles (MSNs) were given a substantial interest due to their unique properties and wide range of applications. The size is a key feature which determines the use of MSNs. Transmission Electron Microscopy (TEM) is a standard technique for MSNs’ size determination. Despite reliable, yet it has some drawbacks, being time consuming, destructive and requires complex sample preparation. Therefore, this work aims at introducing Low-angle X-ray Scattering (LAXS) as a time-saving, non-destructive and inexpensive tool for the determination of the size of MSNs. Different sizes of MSNs are prepared using different concentrations of ammonium hydroxide (catalyst) and their corresponding LAXS profiles are recorded. LAXS profile parameters (peak position and area under peak) are found to exhibit a strong linear dependence on the size of MSNs determined using the standard TEM technique. Moreover, the pore sizes calculated from LAXS profiles show linear dependence on the size of MSNs. For MSNs in the range from 60 to 170 nm, two mathematical linear fitting equations are introduced for the determination of the size of MSNs by direct substitution using LAXS profile parameters. One more equation is introduced for the calculation of the size of MSNs directly from their pore size.