New versatile staining reagents for biological transmission electron microscopy that substitute for uranyl acetate.

Aqueous uranyl acetate has been extensively used as a superb staining reagent for transmission electron microscopy of biological materials. However, recent regulation of nuclear fuel material severely restricts its use even for purely scientific purposes. Since uranyl salts are hazardous due to biological toxicity and remaining radioactivity, development of safe and non-radioactive substitutes is greatly anticipated. We examined two lanthanide salts, samarium triacetate and gadolinium triacetate, and found that 1-10% solution of these reagents was safe but still possess excellent capability for staining thin sections of plastic-embedded materials of animal and plant origin. Although post-fixation with osmium tetroxide was essential for high-contrast staining, post-staining with lead citrate could be eliminated if a slow-scan CCD camera is available for observation. These lanthanide salts can also be utilized as good negative-staining reagents to study supramolecular architecture of biological macromolecules. They were not as effective as a fixative of protein assembly, reflecting the non-hazardous nature of the reagents.

Reducing manual operation time to obtain a segmentation learning model for volume electron microscopy using stepwise deep learning with manual correction.

Three-dimensional (3D) observation of a biological sample using serial-section electron microscopy is widely used. However, organelle segmentation requires a significant amount of manual time. Therefore, several studies have been conducted to improve organelle segmentation's efficiency. One such promising method is 3D deep learning (DL), which is highly accurate. However, the creation of training data for 3D DL still requires manual time and effort. In this study, we developed a highly efficient integrated image segmentation tool that includes stepwise DL with manual correction. The tool has four functions: efficient tracers for annotation, model training/inference for organelle segmentation using a lightweight convolutional neural network, efficient proofreading and model refinement. We applied this tool to increase the training data step by step (stepwise annotation method) to segment the mitochondria in the cells of the cerebral cortex. We found that the stepwise annotation method reduced the manual operation time by one-third compared with the fully manual method, where all the training data were created manually. Moreover, we demonstrated that the F1 score, the metric of segmentation accuracy, was 0.9 by training the 3D DL model with these training data. The stepwise annotation method using this tool and the 3D DL model improved the segmentation efficiency of various organelles.

Degradation of insulin amyloid by antibiotic minocycline and formation of toxic intermediates.

Insulin balls, localized insulin amyloids formed at subcutaneous insulin-injection sites in patients with diabetes, cause poor glycemic control owing to impairments in insulin absorption. Our previous study has shown that some insulin balls are cytotoxic, but others are not, implying amyloid polymorphism. Interestingly, the patient with toxic insulin balls had been treated with antibiotic minocycline, suggesting a possible relationship between toxicity of insulin balls and minocycline. However, the direct effect of minocycline on the structure and cytotoxicity of the insulin amyloid is still unclear. Herein, we demonstrated that that minocycline at physiological concentrations induced degradation of insulin amyloids formed from human insulin and insulin drug preparations used for diabetes patients. Interestingly, the process involved the initial appearance of the toxic species, which subsequently changed into less-toxic species. It is also shown that the structure of the toxic species was similar to that of sonicated fragments of human insulin amyloids. Our study shed new light on the clarification of the revelation of insulin balls and the development of the insulin analogs for diabetes therapy.

Dependence of beam broadening on detection angle in scanning transmission electron microtomography.

It has been shown that scanning transmission electron microtomography (STEMT) is quite effective for observing specimens with thicknesses on the order of micrometers in three dimensions (3D). In STEMT, the specimen is scanned using a focused electron beam, and the electrons from the convergence point are detected at the detector placed at a certain detection angle. Until recently, a wide detection angle corresponding to the mode often called the dark-field (DF) mode was mainly used. Although the detection angle can vary and is one of the crucial experimental factors in STEMT, its effect on 3D reconstruction has never been discussed from either an experimental or a theoretical viewpoint. Moreover, the effectiveness of another mode of electron tomography, transmission electron microtomography (TEMT), is not clear. In the present study, a polymeric specimen, an acrylonitrile butadiene styrene resin, with a thickness of ~1 mum and a fixed volume was observed using three different modes, namely, TEMT, small detection-angle STEMT referred to as bright-field STEMT, and DF-STEMT, in order to examine their advantages and disadvantages by observing multiple scattering of electrons inside the specimen.

Development of novel correlative light and electron microscopy linkage system using silicon nitride film.

In this study, we investigated the optical properties of a silicon nitride (SiN) film. The thin SiN film (30 nm thick) exhibited good light transmittance and little autofluorescence and could be used as a microscope slide for optical microscopy (OM). In addition, we developed a novel correlative light and electron microscopy (CLEM) that combines OM with transmission electron microscopy (TEM) using an SiN thin film. In this system, CLEM was performed by replacing a detachable retainer with a holder for TEM and an adaptor for OM. The advantage of this method is that the same specimens can be sequentially observed using suitable OM and TEM.

Local surface plasmon resonance of gold nanoparticles as a correlative light and electron microscopy (CLEM) tag for biological samples.

In this study, we investigated use of local surface plasmon resonance (LSPR) of metal nanoparticles (NPs) as a correlative light and electron microscopy (CLEM) tag for biological samples. Gold NPs in ultra-thin sections for TEM revealed that LSPR could be observed by optical microscopy at sizes of 20 nm or larger. Gold NPs at sizes less than 20 nm could be observed using the gold enhancement method. Therefore, this CLEM tag could be applied to immunoelectron microscopy using this gold enhancement method.

Practical method of cell segmentation in electron microscope image stack using deep convolutional neural network☆.

Segmentation of three-dimensional (3D) electron microscopy (EM) image stacks is an arduous and tedious task. Deep convolutional neural networks (CNNs) work well to automate the segmentation; however, they require a large training dataset, which is a major impediment. In order to solve this issue, especially for sparse segmentation, we used a CNN with a minimal training dataset. We segmented a Cerebellar Purkinje cell from an image stack of a mouse Cerebellum cortex in less than two working days, which is much shorter than that of the conventional method. We concluded that we can reduce the total labor time for the sparse segmentation by reducing the training dataset.

Observation of the three-dimensional structure of actin bundles formed with polycations.

Three-dimensional structures of actin bundles formed with polycations were observed by using transmission electron microtomography and atomic force microscopy. We found, for the first time, that the cross-sectional morphology of actin bundles depends on the polycation species and ionic strength, while it is insensitive to the degree of polymerization and concentration of polycation. Actin bundles formed with poly-N-[3-(dimethylamino)propyl] acrylamide methyl chloride quaternary show a ribbon-like cross-sectional morphology in low salt concentrations that changes to cylindrical cross-sectional morphology with hexagonal packing of the actin filaments in high salt concentrations. Contrastingly, actin bundles formed with poly-L-lysine show triangular cross-sectional morphology with hexagonal packing of the actin filaments. These variations in cross-sectional morphology are discussed in terms of anisotropy in the electrostatic energy barrier.

Transmission electron microtomography without the "missing wedge" for quantitative structural analysis.

A three-dimensional (3D) visualization and structural analysis of a rod-shaped specimen of a zirconia/polymer nanocomposite material were carried out by transmission electron microtomography (TEMT) with particular emphasis on complete rotation of the specimen (tilt angular range: +/-90 degrees ). In order to achieve such an ideal experimental condition for the TEMT, improvements in the specimen as well as the sample holder were made. A rod-shaped specimen was necessary in order to obtain a high transmission of the specimen upon tilting to large angles. The image resolution of the reconstructed tomogram was isotropic, in sharp contrast to the anisotropic image resolution of the conventional TEMT with a limited angular range (the "missing wedge" problem). A volume fraction of zirconia, phi, evaluated from the 3D reconstruction was in quantitative agreement with the known composition of the nanocomposite. A series of 3D reconstructions was made from the tilt series with complete rotation by limiting the maximum tilt angle, alpha, from which a couple of structural parameters, the volume fraction and surface area per unit volume, Sigma, of the zirconia, were evaluated as a function of alpha. It was confirmed from actual experimental data that both phi and Sigma slightly decreased with the increasing alpha and reached constant values at around alpha=80 degrees , suggesting that the specimen may have to be tilted to +/-80 degrees for truly quantitative measurements.

Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.

Evaluation of lanthanide salts as alternative stains to uranyl acetate.

Uranyl acetate (UAc) has been generally used not only as a superb staining reagent for ultrathin sections of plastic-embedded biological materials, but also as high-contrast negative stains for biological macromolecules such as particles of protein or virus. However, the use and purchase of radioactive UAc have been restricted. In this study, we determine the performance of ytterbium triacetate, lutetium triacetate, samarium triacetate and gadolinium triacetate as new staining reagents for biological electron microscopy. We observed chemically fixed spinach (Spinacia oleracea) leaves stained with these reagents. Ultrathin sections were stained with these reagents. Some of them were counterstained with lead citrate. The transmission electron microscopy contrast of spinach organelles was evaluated in sections exposed to the conventional stain and new stains. We show acetate salts of samarium, gadolinium, ytterbium and lutetium could be excellent substitutes for UAc for thin section staining and for negative staining. In addition, each reagent showed appreciable negative-staining effects.

The spindle pole body of the pathogenic yeast Exophiala dermatitidis: variation in morphology and positional relationship to the nucleolus and the bud in interphase cells.

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Exophiala dermatitidis was studied in detail. The SPB was located on the outer nuclear envelope and was 342 +/- 86 nm long in a haploid strain. It consisted of two disk elements that measured 151 +/- 43 nm in diameter and 103 +/- 17 nm in thickness, connected by a rod-shaped midpiece that measured 56 +/- 20 nm in length and 37 +/- 9 nm in diameter. There were considerable variations in size and morphology of interphase SPB. Some disk elements appeared spherical but others were more flattened, and there was variation in electron density. A few SPBs did not have the midpiece. The SPB of a diploid strain was 486 +/- 118 nm long, thus significantly bigger than that of the haploid strain. The SPB tended to be localized away from the nucleolus (110 +/- 48 degrees), but close to the bud (78 +/- 45 degrees). The present study highlights the necessity of observing a large number of micrographs in three-dimensions to describe accurately the ultrastructure of the SPB in yeast.

The advanced ion-milling method for preparation of thin film using ion slicer: application to a sample recovered from diamond-anvil cell.

The advanced argon ion-milling technique using a new instrument called ion slicer was newly developed for preparation of thin foil. Compared to the conventional ion-milling methods, this technique facilitates very wide area to be homogeneously thinned by rocking the ion beam source with low angle and the specimen during milling. Here we applied this technique to a sample recovered from a laser-heated diamond-anvil cell (DAC). We obtained the thin film of almost entire cross section of the DAC sample along the compression axis, which possesses of approximately (10x50) microm(2). The laser-heated sample is often heterogeneous due to a large temperature gradient. However the chemical analyses are obtained from the whole hot spot under the transmission electron microscope by preparing the thin foil using the ion slicer.

Spatial arrangement of metal nanoparticles supported by porous polymer substrates studied by transmission electron microtomography.

Fine metal particles (nanoparticles) stabilized on porous (polymeric) substrates can be considered as a model system of a high-performance catalyst. In the present study, the substrate was made using the periodic microphase-separated structure of a block copolymer as the template, and the Pd nanoparticles were formed inside the porous material by reduction of the Pd2+ ions with 1-propanol as the reductant. The three-dimensional morphology of such a polymer-Pd hybrid material was studied by transmission electron microtomography. The characteristic structural parameters of the hybrid, e.g., the penetration of the Pd nanoparticles into the polymer substrate, number density of the Pd nanoparticles, and size distribution of the Pd nanoparticles, were measured for the first time.

Reduction of anisotropic image resolution in transmission electron microtomography by use of quadrangular prism-shaped section.

A quadrangular prism specimen (a 'prism-shaped' section) was observed in three-dimension (3-D) by transmission electron microtomography (TEMT) over as wide a tilt range as possible. Two types of specimens were prepared for the TEMT experiments: (i) a prism-shaped section of a block copolymer nanostructure, whose cross section was 200 nm on each side and (ii) a conventional ultrathin section having the same thickness (approximately 200 nm) as the prism-shaped section. Image quality of the projections taken at high tilt angles, e.g. 60 degrees, of the prism-shaped section was considerably better than that of the ultrathin section. This was because the path length of electron beam of the prism-shaped section was shorter (and hence the transmission was higher) than that of the ultrathin section at the same tilt angle. Thus, although the projections of the ultrathin section at the tilt angle larger than approximately 40 degrees did not effectively contribute to the 3-D reconstruction, those of the prism-shaped section at very high tilt angles, e.g. +/-75 degrees, can still be used for the 3-D reconstruction. Three orthogonal cross-sectional views of the 3-D reconstruction were extensively compared between the two sections. It was found that (i) the image contrast of the 3-D reconstruction was significantly enhanced and (ii) an elongation of structural object due to limitation of the angular range in the electron tomography was considerably reduced using the prism-shaped section.