3D Ultrastructure Research Articles

Neural network auto-segmentation of serial-block-face scanning electron microscopy images exhibit collagen fibril structural differences with tendon type and health.

A U-Net machine learning algorithm was adapted to automatically segment tendon collagen fibril cross-sections from serial block face scanning electron microscopy (SBF-SEM) and create three-dimensional (3D) renderings. We compared the performance of routine Otsu thresholding and U-Net for a positional tendon that has low fibril density (rat tail tendon), an energy-storing tendon that has high fibril density (rat plantaris tendon), and a high fibril density tendon hypothesized to have disorganized 3D ultrastructure (degenerated rat plantaris tendon). The area segmentation of the tail and healthy plantaris tendon had excellent accuracy for both the Otsu and U-Net, with an Intersection over Union (IoU) of 0.8. With degeneration, only the U-Net could accurately segment the area, whereas Otsu IoU was only 0.45. For boundary validation, the U-Net outperformed Otsu segmentation for all tendons. The fibril diameter from U-Net was within 10% of the manual segmentation, however, the Otsu underestimated the fibril diameter by 39% in healthy plantaris and by 84% in the degenerated plantaris. Fibril geometry was averaged across the entire image stack and compared across tendon types. The tail had a lower fibril area fraction (58%) and larger fibril diameter (0.31 µm) than the healthy plantaris (67% and 0.21 µm) and degenerated plantaris tendon (66% and 0.19 µm). This method can be applied to a large variety of tissues to quantify 3D collagen fibril structure.

Three-dimensional analysis ofthe intracellular architecture by scanning electron microscopy.

The two-dimensional observation of ultrathin sections from resin-embedded specimens provides an insufficient understanding of the three-dimensional (3D) morphological information of membranous organelles. The osmium maceration method, developed by Professor Tanaka's group >40 years ago, is the only technique that allows direct observation of the 3D ultrastructure of membrane systems using scanning electron microscopy (SEM), without the need for any reconstruction process. With this method, the soluble cytoplasmic proteins are removed from the freeze-cracked surface of cells while preserving the integrity of membranous organelles, achieved by immersing tissues in a diluted osmium solution for several days. By employing the maceration method, researchers using SEM have revealed the 3D ultrastructure of organelles such as the Golgi apparatus, mitochondria and endoplasmic reticulum in various cell types. Recently, we have developed new SEM techniques based on the maceration method to explore further possibilities of this method. These include: (i) a rapid osmium maceration method that reduces the reaction duration of the procedure, (ii) a combination method that combines agarose embedding with osmium maceration to elucidate the 3D ultrastructure of organelles in free and cultured cells and (iii) a correlative immunofluorescence and SEM technique that combines cryosectioning with the osmium maceration method, enabling the correlation of the immunocytochemical localization of molecules with the 3D ultrastructure of organelles. In this paper, we review the novel osmium maceration methods described earlier and discuss their potential and future directions in the field of biology and biomedical research.

Biogenesis, inheritance, and 3D ultrastructure of the microsporidian mitosome.

During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of Encephalitozoon cuniculi (2-12/cell) and Trachipleistophora hominis (14-18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome-mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (Schizosaccharomyces pombe). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).

An intelligent workflow for sub-nanoscale 3D reconstruction of intact synapses from serial section electron tomography

BackgroundAs an extension of electron tomography (ET), serial section electron tomography (serial section ET) aims to align the tomographic images of multiple thick tissue sections together, to break through the volume limitation of the single section and preserve the sub-nanoscale voxel size. It could be applied to reconstruct the intact synapse, which expands about one micrometer and contains nanoscale vesicles. However, there are several drawbacks of the existing serial section ET methods. First, locating and imaging regions of interest (ROIs) in serial sections during the shooting process is time-consuming. Second, the alignment of ET volumes is difficult due to the missing information caused by section cutting and imaging. Here we report a workflow to simplify the acquisition of ROIs in serial sections, automatically align the volume of serial section ET, and semi-automatically reconstruct the target synaptic structure.ResultsWe propose an intelligent workflow to reconstruct the intact synapse with sub-nanometer voxel size. Our workflow includes rapid localization of ROIs in serial sections, automatic alignment, restoration, assembly of serial ET volumes, and semi-automatic target structure segmentation. For the localization and acquisition of ROIs in serial sections, we use affine transformations to calculate their approximate position based on their relative location in orderly placed sections. For the alignment of consecutive ET volumes with significantly distinct appearances, we use multi-scale image feature matching and the elastic with belief propagation (BP-Elastic) algorithm to align them from coarse to fine. For the restoration of the missing information in ET, we first estimate the number of lost images based on the pixel changes of adjacent volumes after alignment. Then, we present a missing information generation network that is appropriate for small-sample of ET volume using pre-training interpolation network and distillation learning. And we use it to generate the missing information to achieve the whole volume reconstruction. For the reconstruction of synaptic ultrastructures, we use a 3D neural network to obtain them quickly. In summary, our workflow can quickly locate and acquire ROIs in serial sections, automatically align, restore, assemble serial sections, and obtain the complete segmentation result of the target structure with minimal manual manipulation. Multiple intact synapses in wild-type rat were reconstructed at a voxel size of 0.664 nm/voxel to demonstrate the effectiveness of our workflow.ConclusionsOur workflow contributes to obtaining intact synaptic structures at the sub-nanometer scale through serial section ET, which contains rapid ROI locating, automatic alignment, volume reconstruction, and semi-automatic synapse reconstruction. We have open-sourced the relevant code in our workflow, so it is easy to apply it to other labs and obtain complete 3D ultrastructures which size is similar to intact synapses with sub-nanometer voxel size.

Rapid in-solution preparation of somatic and meiotic plant cell nuclei for high-quality 3D immunoFISH and immunoFISH-GISH

BackgroundThough multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity.The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH).ResultsThe mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations.ConclusionA simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.

Three dimensional structures of the inner and outer pig petrous bone using FIB-SEM: Implications for development and ancient DNA preservation

We report on the 3D ultrastructure of the mineralized petrous bone of mature pig using focused ion beam – scanning electron microscopy (FIB-SEM). We divide the petrous bone into two zones based on the degree of mineralization; one zone close to the otic chamber has higher mineral density than the second zone further away from the otic chamber. The hypermineralization of the petrous bone results in the collagen D-banding being poorly revealed in the lower mineral density zone (LMD), and absent in the high mineral density zone (HMD). We therefore could not use D-banding to decipher the 3D structure of the collagen assembly. Instead we exploited the anisotropy option in the Dragonfly image processing software to visualize the less mineralized collagen fibrils and/or nanopores that surround the more mineralized zones known as tesselles. This approach therefore indirectly tracks the orientations of the collagen fibrils in the matrix itself. We show that the HMD bone has a structure similar to that of woven bone, and the LMD is composed of lamellar bone with a plywood-like structural motif. This agrees with the fact that the bone close to the otic chamber is fetal bone and is not remodeled. The lamellar structure of the bone further away from the otic chamber is consistent with modeling/remodeling. The absence of the less mineralized collagen fibrils and nanopores resulting from the confluence of the mineral tesselles may contribute to shielding DNA during diagenesis. We show that anisotropy evaluation of the less mineralized collagen fibrils could be a useful tool to analyze bone ultrastructures and in particular the directionality of collagen fibril bundles that make up the bone matrix.

A Three-Dimensional Technique for The Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

Light and electron microscopy continuum-resolution imaging of 3D cell cultures.

3D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tissues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter-scale live-cell light microscopy to nanometer-scale volume electron microscopy by performing 3D cell cultures in a single carrier that is amenable to all imaging steps. This allows for following organoids' growth, probing their morphology with fluorescent markers, identifying areas of interest, and analyzing their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses identify local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum-resolution imaging pipeline is thus suited to fostering basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy.

A single-molecule view on intraflagellar transport in C. elegans chemosensory cilia.

In our cells, motor proteins do not work on their own: cargoes are often transported by multiple motors, of the same type, but often also of opposite directionality. To study motor cooperation, we have been focusing on intraflagellar transport (IFT), which is essential for the assembly and maintenance of cilia. In particular, we study the chemosensory cilia of C. elegans. To visualize IFT with live fluorescence microscopy, we generate mutant worms expressing fluorescent versions of the proteins of interest. Our fluorescence and image-analysis approaches allow us to visualize, track and quantify trains of IFT components moving together, as well as individual motors or IFT proteins. In C. elegans, IFT is driven by groups of tens of three different motor proteins: 2 kinesin-2's (the slow kinesin-II and the faster OSM-3), which drive transport of cargo trains from base to tip of the cilium, and IFT dynein, which drives transport back to the base. Cargo trains move in one go from base to tip and from tip to base. Speed and directionality appear to be regulated by motor proteins docking on and off the trains. Here I present new single-molecule data focusing on the entry of IFT components, including motor proteins, into the cilium. We observe that different IFT components attach to IFT trains in a sequential way, both in time and in location. Pooling the localization information of many individual molecules allows mapping of the 3D ultrastructure of the axoneme with a precision of several tens of nanometers, in wild-type as well as mutant strains lacking e.g. motors. These maps provide new insights in ciliary structure at the base, train-docking dynamics and the interplay of kinesin-II and OSM-3. Our results provide important new insights into how motors cooperate to drive intracellular transport.

Scaffold-based tissue engineering: Supercritical carbon dioxide as an alternative method for decellularization and sterilization of dense materials.

Development of ready-to-use biomaterials and scaffolds is vital for further advancement of scaffold-based tissue engineering in clinical practice. Scaffolds need to mimic 3D ultrastructure, have adequate mechanical strength, are biocompatible, non-immunogenic and need to promote tissue regeneration in vivo. Although decellularization of native tissues seems promising to deliver scaffolds that meet these criteria, adequate decellularization of hard, poorly penetrable and poorly diffusible tissues remains challenging whilst being a very time-consuming process. In this study, a method to decellularize hard, dense tissues using supercritical carbon-dioxide preceded by a freeze/thaw cycle and followed by several washing steps is presented, demonstrating decellularisation efficiency and substantially reduced production/handling time. Additionally, supercritical carbon-dioxide treatment was used as sterilization method, further reducing the time required to produce the final scaffold. Histological evaluation showed that, after fine-tuning of the process, a partially acellular scaffold was obtained, with preservation of glycosaminoglycans and collagen fibers, albeit that the amount of residual dsDNA was still higher then chemically decellularized tissue. Biomechanical properties of the scaffold were similar to the native, non-decellularized tissue. After sterilization with supercritical carbon-dioxide the simulated functional outcome was more similar to native trachea, when compared to sterilization using gamma irradiation. Thus, decellularization and sterilization using supercritical carbon-dioxide with washing steps is an effective method for dense cartilaginous materials, and tuneable to meet different demands in other applications, but further optimization may be required. STATEMENT OF SIGNIFICANCE: Further advancement of the use of tissue engineered tracheal constructs is restricted by the lack of the ideal scaffold. Decellularized trachea is considered a promising scaffold, but the hard, poorly diffusible tissue remains challenging while forming a very time consumable process. Decellularization using supercritical carbon dioxide (scCO2) seems promising, resulting in efficient removal of cellular material while reducing production and handling time. Addition of scCO2 as a sterilization method resulted in further time reduction while improving functional outcome in comparison with traditional sterilization methods. This study presents an promising alternative method for decellularization and sterilization of dense materials, which can be tuned to meet different demands in other applications.

The 3D ultrastructure of the chordotonal organs in the antenna of a microwasp remains complex although simplified

Insect antennae are astonishingly versatile and have multiple sensory modalities. Audition, detection of airflow, and graviception are combined in the antennal chordotonal organs. The miniaturization of these complex multisensory organs has never been investigated. Here we present a comprehensive study of the structure and scaling of the antennal chordotonal organs of the extremely miniaturized parasitoid wasp Megaphragma viggianii based on 3D electron microscopy. Johnston’s organ of M. viggianii consists of 19 amphinematic scolopidia (95 cells); the central organ consists of five scolopidia (20 cells). Plesiomorphic composition includes one accessory cell per scolopidium, but in M. viggianii this ratio is only 0.3. Scolopale rods in Johnston’s organ have a unique structure. Allometric analyses demonstrate the effects of scaling on the antennal chordotonal organs in insects. Our results not only shed light on the universal principles of miniaturization of sense organs, but also provide context for future interpretation of the M. viggianii connectome.

Synaptic spinules are reliable indicators of excitatory presynaptic bouton size and strength and are ubiquitous components of excitatory synapses in CA1 hippocampus.

Synaptic spinules are thin, finger-like projections from one neuron that become embedded within the presynaptic or postsynaptic compartments of another neuron. While spinules are conserved features of synapses across the animal kingdom, their specific function(s) remain unknown. Recent focused ion beam scanning electron microscopy (FIB-SEM) image volume analyses have demonstrated that spinules are embedded within ∼25% of excitatory boutons in primary visual cortex, yet the diversity of spinule sizes, origins, and ultrastructural relationships to their boutons remained unclear. To begin to uncover the function of synaptic spinules, we sought to determine the abundance, origins, and 3D ultrastructure of spinules within excitatory presynaptic spinule-bearing boutons (SBBs) in mammalian CA1 hippocampus and compare them with presynaptic boutons bereft of spinules (non-SBBs). Accordingly, we performed a comprehensive 3D analysis of every excitatory presynaptic bouton, their embedded spinules, and postsynaptic densities, within a 5 nm isotropic FIB-SEM image volume from CA1 hippocampus of an adult male rat. Surprisingly, we found that ∼74% of excitatory presynaptic boutons in this volume contained at least one spinule, suggesting they are fundamental components of excitatory synapses in CA1. In addition, we found that SBBs are 2.5-times larger and have 60% larger postsynaptic densities (PSDs) than non-SBBs. Moreover, synaptic spinules within SBBs are clearly differentiated into two groups: small clathrin-coated spinules, and 29-times larger spinules without clathrin. Together, these findings suggest that the presence of a spinule is a marker for stronger and more stable presynaptic boutons in CA1, and that synaptic spinules serve at least two separable and distinct functions.

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy.

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

Three-dimensional analysis of interstitial cells in the lamina propria of the murine vas deferens by confocal laser scanning microscopy and FIB/SEM.

The present study aimed to explore the three-dimensional (3D) ultrastructure of interstitial cells (ICs) within the lamina propria of the murine vas deferens and the spatial relationships between epithelial cells and surrounding cells. Focused ion beam scanning electron microscopy and confocal laser scanning microscopy were performed. ICs within the lamina propria had a flat, sheet-like structure of cytoplasm with multiple cellular processes. In addition, two types of 3D structures that comprised cell processes of flat, sheet-like ICs were observed: one was an accordion fold-like structure and the other was a rod-shaped structure. ICs were located parallel to the epithelium and were connected to each other via gap junctions or adherens junctions. Moreover, multiple sphere-shaped extracellular vesicle-like structures were frequently observed around the ICs. The ICs formed a complex 3D network comprising sheet-like cytoplasm and multiple cell processes with different 3D structures. From this morphological study, we noted that ICs within the lamina propria of murine vas deferens may be involved in signal transmission between the epithelium and smooth muscle cells by physical interaction and by exchanging extracellular vesicles.

Combined use of serial block face SEM and focused ion beam SEM elucidates the 3D ultrastructure of blood platelets and thrombi

With the emergence of the 3D electron microscopy techniques, serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), it has become feasible to image cellular and tissue ultrastructure at a nanoscale. In the field of homeostasis and thrombosis, both techniques enable investigation of platelet activation and clotting. We find that FIB-SEM provides 3D ultrastructure from volumes in the range 10,000 to 100,000 cubic micrometers, revealing alpha-granules and the canalicular system at a near-isotropic spatial resolution of ∼5 nm.

Three-Dimensional Analysis of Interstitial Cells in the Smooth Muscle Layer of Murine Vas Deferens Using Confocal Laser Scanning Microscopy and FIB/SEM.

The smooth muscle contraction of the vas deferens has the important function of transporting sperm. Interstitial cells (ICs) play a critical role in the pacing and modulation of various smooth muscle organs by interactions with nerves and smooth muscle. Elucidating the three-dimensional (3D) architecture of ICs is important for understanding their spatial relationship on the mesoscale between ICs, smooth muscle cells (SMCs), and nerves. In this study, the 3D ultrastructure of ICs in the smooth muscle layer of murine vas deferens and the spatial relationships between ICs, nerves, and smooth muscles were observed using confocal laser scanning microscopy and focused ion beam/scanning electron microscopy. ICs have sheet-like structures as demonstrated by 3D observation using modern analytical techniques. Sheet-like ICs have two types of 3D structures, one flattened and the other curled. Multiple extracellular vesicle (EV)-like structures were frequently observed in ICs. Various spatial relations were observed in areas between ICs, nerves, and SMCs, which formed a complex 3D network with each other. These results suggest that ICs in the smooth muscle layer of murine vas deferens may have two subtypes with different sheet-like structures and may be involved in neuromuscular signal transmission via physical interaction and EVs.

Spermidine supplementation influences mitochondrial number and morphology in the heart of aged mice.

Aging is associated with cardiac hypertrophy and progressive decline in heart function. One of the hallmarks of cellular aging is the dysfunction of mitochondria. These organelles occupy around 1/4 to 1/3 of the cardiomyocyte volume. During cardiac aging, the removal of defective or dysfunctional mitochondria by mitophagy as well as the dynamic equilibrium between mitochondrial fusion and fission is distorted. Here, we hypothesized that these changes affect the number of mitochondria and alter their three-dimensional (3D) characteristics in aged mouse hearts. The polyamine spermidine stimulates both mitophagy and mitochondrial biogenesis, and these are associated with improved cardiac function and prolonged lifespan. Therefore, we speculated that oral spermidine administration normalizes the number of mitochondria and their 3D morphology in aged myocardium. Young (4-months old) and old (24-months old) mice, treated or not treated with spermidine, were used in this study (n=10 each). The number of mitochondria in the left ventricles was estimated by design-based stereology using the Euler-Poincaré characteristic based on a disector at the transmission electron microscopic level. The 3D morphology of mitochondria was investigated by 3D reconstruction (using manual contour drawing) from electron microscopic z-stacks obtained by focused ion beam scanning electron microscopy. The volume of the left ventricle and cardiomyocytes were significantly increased in aged mice with or without spermidine treatment. Although the number of mitochondria was similar in young and old control mice, it was significantly increased in aged mice treated with spermidine. The interfibrillar mitochondria from old mice exhibited a lower degree of organization and a greater variation in shape and size compared to young animals. The mitochondrial alignment along the myofibrils in the spermidine-treated mice appeared more regular than in control aged mice, however, old mitochondria from animals fed spermidine also showed a greater diversity of shape and size than young mitochondria. In conclusion, mitochondria of the aged mouse left ventricle exhibited changes in number and 3D ultrastructure that is likely the structural correlate of dysfunctional mitochondrial dynamics. Spermidine treatment reduced, at least in part, these morphological changes, indicating a beneficial effect on cardiac mitochondrial alterations associated with aging.

Applications of Scanning Electron Microscopy Using Secondary and Backscattered Electron Signals in Neural Structure.

Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography (i.e., serial section SEM). This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods.

Tissue engineering of mouse uterus using menstrual blood stem cells (MenSCs) and decellularized uterine scaffold

BackgroundUterine tissue engineering can provide the opportunity for curing female infertility. Natural scaffold is a good choice to recapitulate the architecture and functionality of the native tissue. In this study, we purposed the potential of uterine decellularized scaffolds as an adequate natural niche for MenSCs differentiation toward uterus-specific cell lineages.MethodsMouse’s uterus was decellularized by immersion of hypo and hypertonic salts or freeze–thaw cycle followed by immersion in Triton X-100 and SDS solutions. MenSCs were isolated from the menstrual blood of 6 healthy women. The decellularized and recellularized samples were prepared for further in vitro and in vivo analyses.ResultsHistochemical studies and Raman spectroscopy revealed uterine ECM was preserved well, and the cells were completely removed after decellularization. Scanning electron microscopy (SEM) showed that the 3D ultrastructure of the uterus remained intact. Flowcytometric examination with CD34, CD44, CD105, CD106, CD144 markers revealed stem cell characters of cells that isolated from menstrual blood. MTT assay confirmed the bioavailability of MenSCs cultured scaffolds after 7 and 10 days.ConclusionHistochemical studies, SEM images, and Raman spectra showed MenSCs seeded and growth in uterine scaffolds. Immunostaining using anti-cytokeratin (CK), anti-desmin (Des), anti-vimentin (Vim), and anti-smooth muscle actin (SMA) antibodies showed MenSCs differentiation to epithelial and smooth muscle tissues. The Raman spectroscopy revealed the extracellular matrix (ECM) of decellularized uterine scaffolds was well preserved. The decellularized uterus can be considered a promising vehicle to support cell transplantation and differentiation. MenSCs are a good choice for uterine tissue engineering. The complete decellularization from mice uterine tissue was done by combining chemical agents

Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells

Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked “density scissors” connect with one another to form a “scissors stack network (SSN)” plane oriented 45° to the axoneme axis; and ~370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with ~90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics.