Particle Size Measurement and Detection of Bound Proteins of Non-Porous/Mesoporous Silica Microspheres by Single-Particle Inductively Coupled Plasma Mass Spectrometry.

Single-particle inductively coupled plasma mass spectrometry (spICP-MS) has been used for particle size measurement of diverse types of individual nanoparticles and micrometer-sized carbon-based particles such as microplastics. However, its applicability to the measurement of micrometer-sized non-carbon-based particles such as silica (SiO2) particles is unclear. In this study, the applicability of spICP-MS to particle size measurement of non-porous/mesoporous SiO2 microspheres with a nominal diameter of 5.0 µm or smaller was investigated. Particle sizes of these microspheres were measured using both spICP-MS based on a conventional calibration approach using an ion standard solution and scanning electron microscopy as a reference technique, and the results were compared. The particle size distributions obtained using both techniques were in agreement within analytical uncertainty. The applicability of this technique to the detection of metal-containing protein-binding mesoporous SiO2 microspheres was also investigated. Bound iron (Fe)-containing proteins (i.e., lactoferrin and transferrin) of mesoporous SiO2 microspheres were detected using Fe as a presence marker for the proteins. Thus, spICP-MS is applicable to the particle size measurement of large-sized and non-porous/mesoporous SiO2 microspheres. It has considerable potential for element-based detection and qualification of bound proteins of mesoporous SiO2 microspheres in a variety of applications.

Direct observation of the effects of chemical fixation in MNT-1 cells: A SE-ADM and Raman study.

Aldehydes fixation was accidentally discovered in the early 20th century and soon became a widely adopted practice in the histological field, due to an excellent staining enhancement in tissues imaging. However, the fixation process itself entails cell proteins denaturation and crosslinking. The possible presence of artifacts, that depends on the specific system under observation, must therefore be considered to avoid data misinterpretation. This contribution takes advantage of scanning electron assisted-dielectric microscopy (SE-ADM) and Raman 2D imaging to reveal the possible presence and the nature of artifacts in unstained, and paraformldehyde, PFA, fixed MNT-1 cells. The high resolution of the innovative SE-ADM technique allowed the identification of globular protein clusters in the cell cytoplasm, formed after protein denaturation and crosslinking. Concurrently, SE-ADM images showed a preferential melanosome adsorption on the cluster's outer surface. The micron-sized aggregates were discernible in Raman 2D images, as the melanosomes signal, extracted through 2D principal component analysis, unequivocally mapped their location and distribution within the cells, appearing randomly distributed in the cytoplasm. Protein clusters were not observed in living MNT-1 cells. In this case, mature melanosomes accumulate preferentially at the cell periphery and are more closely packed than in fixed cells. Our results show that, although PFA does not affect the melanin structure, it disrupts melanosome distribution within the cells. Proteins secondary structure, conversely, is partially lost, as shown by the Raman signals related to α-helix, ß-sheets, and specific amino acids that significantly decrease after the PFA treatment.

Development of General-purpose Dielectric Constant Imaging Unit for SEM and Direct Observation of Samples in Aqueous Solution.

Electron microscopes can observe samples with a spatial resolution of 10 nm or higher; however, they cannot observe samples in solutions due to the vacuum conditions inside the sample chamber. Recently, we developed a scanning electron-assisted dielectric microscope (SE-ADM), based on scanning electron microscope, which enables the observation of various specimens in solution. Until now, the SE-ADM system used a custom-made SE-ADM stage with a built-in amplifier and could not be linked to the scanning electron microscopy (SEM) operation system. Therefore, it was necessary to manually acquire images from the SE-ADM system after setting the EB focus, astigmatism, and observation field-of-view from the SEM operating console. In this study, we developed a general-purpose dielectric constant imaging unit attached to commercially available SEMs. The new SE-ADM unit can be directly attached to the standard stage of an SEM, and the dielectric signal detected from this unit can be input to the external input terminal of the SEM, enabling simultaneous observation yielding SEM and SE-ADM images. Furthermore, 4.5 nm spatial resolution was achieved using a 10 nm thick silicon nitride film in the sample holder in the observation of aggregated PM2.5. We carried out the observation of cultured cells, PM2.5, and clay samples in solution.

Surface light propagating in a dielectric thin film generated via micro-spheres.

Light orbiting through total internal reflection within dielectric spheres or disks is called the whispering gallery mode (WGM). Recently, we have reported anomalously enhanced Raman spectra at the periphery of 3 µm diameter polystyrene (PS) microspheres on a silicon nitride (SiN) film using Raman microscopy. Here, we performed Raman measurements and optical simulation analysis of 3 µm PS spheres on a SiN film using a three-dimensional (3D) model and found that the circumferential light was generated up to 650 nm from the outer circumference of the sphere. Furthermore, a portion of the light circling the sphere travelled to the SiN film and became surface propagating light. These properties are expected to lead to development of new devices such as highly sensitive sensors, quantum optical qubits, and optical integrated circuits.

Reversible adsorption of iridium in lyophilized cells of the unicellular red alga Galdieria sulphuraria.

Iridium (Ir) is one of the rarest elements in the Earth's crust and is valuable in industry due to its high corrosion resistance. In this study, we used lyophilized cells of a unicellular red alga, Galdieria sulphuraria for the selective recovery of small amounts of Ir from hydrochloric acid (HCl) solutions. The Ir recovery efficiency of the lyophilized cells was higher than that of activated carbon and comparable to that of an ion-exchange resin in up to 0.2 M acid. Lyophilized G. sulphuraria cells showed different selectivity from the ion-exchange resin, adsorbing Ir and Fe in 0.2 M HCl solution while the ion-exchange resin adsorbed Ir and Cd. The adsorbed Ir could be eluted with more than 90% efficiency using HCl, ethylenediaminetetraacetic acid, and potassium hydroxide solutions, but could not be eluted using a thiourea-HCl solution. After the elution of Ir with a 6 M HCl solution, lyophilized cells could be reused up to five times for Ir recovery with over 60% efficiency. Scanning electron-assisted dielectric microscopy and scanning electron microscopy revealed that Ir accumulated in the cytosol of the lyophilized cells. X-ray absorption fine structure analysis demonstrated the formation of an outer-sphere complex between Ir and the cellular residues, suggesting the adsorption via ion exchange, and explaining the ability to elute the Ir and reuse the cells. Our results provide a scientific basis for inexpensive and environmentally friendly biosorbents as an alternative to ion-exchange resins for the recovery of Ir.

Structural analysis of melanosomes in living mammalian cells using scanning electron-assisted dielectric microscopy with deep neural network.

Melanins are the main pigments found in mammals. Their synthesis and transfer to keratinocytes have been widely investigated for many years. However, analysis has been mainly carried out using fixed rather than live cells. In this study, we have analysed the melanosomes in living mammalian cells using newly developed scanning electron-assisted dielectric microscopy (SE-ADM). The melanosomes in human melanoma MNT-1 cells were observed as clear black particles in SE-ADM. The main structure of melanosomes was toroidal while that of normal melanocytes was ellipsoidal. In tyrosinase knockout MNT-1 cells, not only the black particles in the SE-ADM images but also the Raman shift of melanin peaks completely disappeared suggesting that the black particles were really melanosomes. We developed a deep neural network (DNN) system to automatically detect melanosomes in cells and analysed their diameter and roundness. In terms of melanosome morphology, the diameter of melanosomes in melanoma cells did not change while that in normal melanocytes increased during culture. The established DNN analysis system with SE-ADM can be used for other particles, e.g. exosomes, lysosomes, and other biological particles.

Spermidine activates mitochondrial trifunctional protein and improves antitumor immunity in mice.

Spermidine (SPD) delays age-related pathologies in various organisms. SPD supplementation overcame the impaired immunotherapy against tumors in aged mice by increasing mitochondrial function and activating CD8+ T cells. Treatment of naïve CD8+ T cells with SPD acutely enhanced fatty acid oxidation. SPD conjugated to beads bound to the mitochondrial trifunctional protein (MTP). In the MTP complex, synthesized and purified from Escherichia coli, SPD bound to the α and ß subunits of MTP with strong affinity and allosterically enhanced their enzymatic activities. T cell-specific deletion of the MTP α subunit abolished enhancement of programmed cell death protein 1 (PD-1) blockade immunotherapy by SPD, indicating that MTP is required for SPD-dependent T cell activation.

Giant Carbon Nano-Test Tubes as Versatile Imaging Vessels for High-Resolution and In Situ Observation of Proteins.

Cryogenic electron microscopy is one of the fastest and most robust methods for capturing high-resolution images of proteins, but stringent sample preparation, imaging conditions, and in situ radiation damage inflicted during data acquisition directly affect the resolution and ability to capture dynamic details, thereby limiting its broader utilization and adoption for protein studies. We addressed these drawbacks by introducing synthesized giant carbon nano-test tubes (GCNTTs) as radiation-insulating materials that lessen the irradiation impact on the protein during data acquisition, physical molecular concentrators that localize the proteins within a nanoscale field of view, and vessels that create a microenvironment for solution-phase imaging. High-resolution electron microscopy images of single and aggregated hemoglobin molecules within GCNTTs in both solid and solution states were acquired. Subsequent scanning transmission electron microscopy, small-angle neutron scattering, and fluorescence studies demonstrated that the GCNTT vessel protected the hemoglobin molecules from electron irradiation-, light-, or heat-induced denaturation. To demonstrate the robustness of GCNTT as an imaging platform that could potentially augment the study of proteins, we demonstrated the robustness of the GCNTT technique to image an alternative protein, d-fructose dehydrogenase, after cyclic voltammetry experiments to review encapsulation and binding insights. Given the simplicity of the material synthesis, sample preparation, and imaging technique, GCNTT is a promising imaging companion for high-resolution, single, and dynamic protein studies under electron microscopy.

The therapeutic capacity of bone marrow MSC-derived extracellular vesicles in Achilles tendon healing is passage-dependent and indicated by specific glycans.

The therapeutic potential of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) for various diseases and tissue repair is attracting attention. Here, EVs from conditioned medium of human bone marrow MSCs at passage 5 (P5) and passage 12 (P12) were analysed using mouse Achilles tendon rupture model and lectin microarray. P5 MSC-EVs accelerated Achilles tendon healing compared with P12 MSC-EVs. Fucose-specific lectin TJA-II was indicated as a glycan marker for therapeutic MSC-EVs. The present study demonstrated that early passaged MSC-EVs promote Achilles tendon healing compared with senescent MSC-EVs. Glycans on MSC-EVs might provide useful tools to establish a quality control and isolation system for therapeutic MSC-EVs in regenerative medicine.

Raman scattering enhancement of dielectric microspheres on silicon nitride film.

Circulating light in the total internal reflection within dielectric spheres or disks is called the whispering gallery mode (WGM), which by itself is highly sensitive to its surface and capable of detecting viruses and single atomic ions. The detection site of the sensors using WGM is created by the evanescent light from the circulating light inside spheres. Here we report anomalous Raman scattering enhancement in dielectric microspheres on a silicon nitride (SiN) film. This Raman enhancement occurs at the periphery of the spheres, and a similar ring of light was also observed under a fluorescence microscope. This is caused by the light circulating around the dielectric spheres as in the WGM. We observed anomalously enhanced Raman spectrum at the periphery of 3 µm diameter polystyrene (PS) microspheres on a SiN film using confocal laser Raman microscopy. The wavelength intensity of this enhanced Raman spectrum was accompanied by periodic changes due to interference. These features may lead to the development of high-sensitive sensors and optical devices.

Opalescence Arising from Network Assembly in Antibody Solution.

Opalescence of therapeutic antibody solutions is one of the concerns in drug formulation. However, the mechanistic insights into the opalescence of antibody solutions remain unclear. Here, we investigated the assembly states of antibody molecules as a function of antibody concentration. The solutions of bovine gamma globulin and human immunoglobulin G at around 100 mg/mL showed the formation of submicron-scale network assemblies. The network assembly resulted in the appearance of opalescence with a transparent blue color without the precipitates of antibodies. Furthermore, the addition of trehalose and arginine, previously known to act as protein stabilizers and protein aggregation suppressors, was able to suppress the opalescence arising from the network assembly. These results will provide an important information for evaluating and improving protein formulations.

Development of multi-frequency impedance scanning electron microscopy.

Nanometre-scale observation of specimens in water is indispensable in many scientific fields like biology, chemistry, material science and nanotechnology. Scanning electron microscopy (SEM) allows high-resolution images of biological samples to be obtained under high vacuum conditions but requires specific sample-preparation protocols. Therefore, there is a need for convenient and minimally invasive methods of observing samples in solution. We have developed a new type of impedance microscopy, namely multi-frequency impedance SEM (IP-SEM), which allows nanoscale imaging of various specimens in water while minimising radiation damage. By varying the frequency of the input voltage signal of the sine wave, the present system can detect dielectric properties of the sample's composition at nanometre resolution. It also enables examination of unstained biological specimens and material samples in water. Furthermore, it can be used for diverse samples in liquids across a broad range of scientific subjects such as nanoparticles, nanotubes and organic and catalytic materials.

Recovery of Au from dilute aqua regia solutions via adsorption on the lyophilized cells of a unicellular red alga Galdieria sulphuraria: A mechanism study.

The high electrical conductivity, chemical stability, and low toxicity of elemental Au make it a highly valuable resource. However, wastewater produced during the mining, utilization, and disposal of Au inevitably contains small amounts (10-40 mg L-1) of Au, thus posing environmental risks. It is too acidic to be treated with inexpensive and eco-friendly bioadsorbents previously studied for the remediation of less acidic effluents. Herein, lyophilized Galdieria sulphuraria cells are shown to directly adsorb Au from simulated Au-containing wastewater with a total acid concentration of 4 M, achieving an adsorption capacity of 35 ± 2.5 mg g-1 Au after 30-min exposure and a selectivity that exceeds that of an ion-exchange resin and is comparable to that of activated carbon. Additionally, Au adsorbed on these cells is more easily eluted than that adsorbed on the ion-exchange resin or activated carbon. Detailed characterizations reveal that Au accumulates on the surface of lyophilized cells, where it is mainly present as AuCl4- and not as Au0, in contrast to a previously proposed adsorption mechanism. Thus, our work provides valuable insights into the mechanism of Au adsorption on biomaterials and paves the way to the cheap and eco-friendly recovery of Au from acidic wastewater.

Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution.

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid-liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.

Nano-Microscopy of Therapeutic Antibody Aggregates in Solution.

Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.

Findings from recent studies by the Japan Aerospace Exploration Agency examining musculoskeletal atrophy in space and on Earth.

The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly.

Direct Observation of the Relationship between Thixotropic Behavior and Shear-Induced Orientation of Clay Particles in Synesthetic Hectorite Suspensions.

A thixotropic characteristics of aqueous gels containing smectite clay minerals were used in various industrial applications such as paint additives, which have been affected by the clay types and clay particle sizes. A model called a house-of-card arrangement of clay particles and anisotropic arrangement in aqueous gels has been proposed. We prepared different sizes of synthetic hectorite and studied them by scanning electron-assisted dielectric microscopy (SE-ADM) and simultaneous small-angle neutron scattering and rheological measurements (Rheo-SANS). The Rheo-SANS results indicated that the clay particles with the cross-sectional radius of 30 nm were clearly oriented in the direction of shear-flow (1 × 103 s-1) direction, but the anisotropic change was not observed for an aqueous gel with clays whose average radius was 19.5 nm. The present study suggested the thixotropic characteristics of aqueous gels depend on the hectorite particle size and aggregation structure under shear conditions.

Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells.

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

Structural insights into vesicle amine transport-1 (VAT-1) as a member of the NADPH-dependent quinone oxidoreductase family.

Vesicle amine transport protein-1 (VAT-1) has been implicated in the regulation of vesicular transport, mitochondrial fusion, phospholipid transport and cell migration, and is a potential target of anticancer drugs. Little is known about the molecular function of VAT-1. The amino acid sequence indicates that VAT-1 belongs to the quinone oxidoreductase subfamily, suggesting that VAT-1 may possess enzymatic activity in unknown redox processes. To clarify the molecular function of VAT-1, we determined the three-dimensional structure of human VAT-1 in the free state at 2.3 Å resolution and found that VAT-1 forms a dimer with the conserved NADPH-binding cleft on each protomer. We also determined the structure of VAT-1 in the NADP-bound state at 2.6 Å resolution and found that NADP binds the binding cleft to create a putative active site with the nicotine ring. Substrate screening suggested that VAT-1 possesses oxidoreductase activity against quinones such as 1,2-naphthoquinone and 9,10-phenanthrenequinone.

Nanoscale observation of PM2.5 incorporated into mammalian cells using scanning electron-assisted dielectric microscope.

PM2.5 has been correlated with risk factors for various diseases and infections. It promotes tissue injury by direct effects of particle components. However, effects of PM2.5 on cells have not been fully investigated. Recently, we developed a novel imaging technology, scanning electron-assisted dielectric-impedance microscopy (SE-ADM), which enables observation of various biological specimens in aqueous solution. In this study, we successfully observed PM2.5 incorporated into living mammalian cells in culture media. Our system directly revealed the process of PM2.5 aggregation in the cells at a nanometre resolution. Further, we found that the PM2.5 aggregates in the intact cells were surrounded by intracellular membrane-like structures of low-density in the SE-ADM images. Moreover, the PM2.5 aggregates were shown by confocal Raman microscopy to be located inside the cells rather than on the cell surface. We expect our method to be applicable to the observation of various nanoparticles inside cells in culture media.