Insights on Platelet-Derived Growth Factor Receptor α-Positive Interstitial Cells in the Male Reproductive Tract.

Male infertility is a significant factor in approximately half of all infertility cases and is marked by a decreased sperm count and motility. A decreased sperm count is caused by not only a decreased production of sperm but also decreased numbers successfully passing through the male reproductive tract. Smooth muscle movement may play an important role in sperm transport in the male reproductive tract; thus, understanding the mechanism of this movement is necessary to elucidate the cause of sperm transport disorder. Recent studies have highlighted the presence of platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PICs) in various smooth muscle organs. Although research is ongoing, PICs in the male reproductive tract may be involved in the regulation of smooth muscle movement, as they are in other smooth muscle organs. This review summarizes the findings to date on PICs in male reproductive organs. Further exploration of the structural, functional, and molecular characteristics of PICs could provide valuable insights into the pathogenesis of male infertility and potentially lead to new therapeutic approaches.

Three-dimensional ultrastructural and anatomical analysis of prostatic neuroendocrine cells in mice.

BACKGROUND: A few studies have examined the ultrastructure of prostatic neuroendocrine cells (NECs), and no study has focused on their ultrastructure in three dimensions. In this study, three-dimensional ultrastructural analysis of mouse prostatic NECs was performed to clarify their anatomical characteristics. METHODS: Three 13-week-old male C57BL/6 mice were deeply anesthetized, perfused with physiological saline and 2% paraformaldehyde, and then placed in 2.5% glutaraldehyde in 0.1 M cacodylate (pH 7.3) buffer for electron microscopy. After perfusion, the lower urinary tract, which included the bladder, prostate, coagulation gland, seminal vesicle, upper vas deferens, and urethra, was removed, and the specimen was cut into small cubes and subjected to postfixation and en bloc staining. Three-dimensional ultrastructural analysis was performed on NECs, the surrounding cells, tissues, and nerves using focused ion beam/scanning electron microscope tomography. RESULTS: Twenty-seven serial sections were used in the present study, and 32 mouse prostatic NECs were analyzed. Morphologically, the NECs could be classified into three types: flask, flat, and closed. Closed-shaped NECs were always adjacent to flask-shaped cells. The flask-shaped and flat NECs were in direct contact with the ductal lumen and always had microvilli at their contact points. Many of the NECs had accompanying nerves, some of which terminated on the surface in contact with the NEC. CONCLUSIONS: Three-dimensional ultrastructural analysis of mouse prostatic NECs was performed. These cells can be classified into three types based on shape. Novel findings include the presence of microvilli at their points of contact with the ductal lumen and the presence of accompanying nerves.

Sheet-like interstitial cells connect epithelial and smooth muscle cells in the mouse prostate.

Epithelial-stromal relationship in the prostate gland is crucial for maintaining homeostasis, including functional differentiation, proliferation, and quiescence. Pathological stromal changes are believed to cause benign prostatic hyperplasia (BPH). The prostate stromal tissue is known to have several subtypes of interstitial cells that connect the epithelium and smooth muscle. However, the characteristics of their morphology and connection patterns are not fully understood. Therefore, we aimed to investigated the three-dimensional morphology and intercellular interactions of interstitial cells in the prostate ventral lobe of mature wild-type mice using immunohistochemistry and focused ion beam-scanning electron microscopy tomography (FIB-SEM tomography). The prostate interstitial cells exhibited immunohistochemical subtypes, including PDGFRα single-positive, CD34 single-positive, and CD34 and PDGFRα double-positive. PDGFRα single-positive cells were observed as elongated cells just below the epithelium, CD34 single-positive cells were observed as polygonal cells in the area away from the epithelium, and double-positive cells were observed as elongated cells situated slightly deeper than PDGFRα single-positive cells. Furthermore, connexin43-immunoreactive puncta were observed on interstitial cells just beneath the epithelium, suggestive of possible electrical connections among the PDGFRα single-positive interstitial cells. Three-dimensional structural analysis using FIB-SEM tomography revealed sheet-like multilayered interstitial cells that appear to separate the glandular terminal from the deeper interstitial tissue, which includes smooth muscle and capillaries. Further, epithelial cells might be indirectly connected to the smooth muscle and nerve fibers via these sheet-like multilayered interstitial cellular networks. These findings suggest that the cellular network that separates the glandular terminals from the deep interstitial tissue functionally bridges the epithelium and smooth muscle, possibly playing a pivotal role in prostate tissue homeostasis through the epithelial-smooth muscle or epithelial-stromal relationships.

Three-dimensional architecture and assembly mechanism of the egg-shaped shell in testate amoeba Paulinella micropora.

Unicellular euglyphid testate amoeba Paulinella micropora with filose pseudopodia secrete approximately 50 siliceous scales into the extracellular template-free space to construct a shell isomorphic to that of its mother cell. This shell-constructing behavior is analogous to building a house with bricks, and a complex mechanism is expected to be involved for a single-celled amoeba to achieve such a phenomenon; however, the three-dimensional (3D) structure of the shell and its assembly in P. micropora are still unknown. In this study, we aimed to clarify the positional relationship between the cytoplasmic and extracellular scales and the structure of the egg-shaped shell in P. micropora during shell construction using focused ion beam scanning electron microscopy (FIB-SEM). 3D reconstruction revealed an extensive invasion of the electron-dense cytoplasm between the long sides of the positioned and stacked scales, which was predicted to be mediated by actin filament extension. To investigate the architecture of the shell of P. micropora, each scale was individually segmented, and the position of its centroid was plotted. The scales were arranged in a left-handed, single-circular ellipse in a twisted arrangement. In addition, we 3D printed individual scales and assembled them, revealing new features of the shell assembly mechanism of P. micropora. Our results indicate that the shell of P. micropora forms an egg shape by the regular stacking of precisely designed scales, and that the cytoskeleton is involved in the construction process.

Three-Dimensional Ultrastructural and Volume Analysis of the Redundant Nuclear Envelope of Developing and Matured Sperm in Mice.

The ultrastructure of the nuclear envelope (NE) and redundant NE (RNE) of the spermatozoon cannot be observed in detail using conventional electron microscopy. Thus, this study aimed to employ transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography to fill this research gap. Male mice aged 13 weeks were deeply anesthetized, and the testes and vas deferens were extracted and processed for electron microscopy. In round spermatids, the acrosomal vesicle compressed the nucleus, and the acrosomal center was depressed. The nucleoli concentrated on the contralateral side of the acrosome formation site. In mature spermatozoa, the RNE accumulated in the neck with the residual bodies. The NE pores exhibited a hexagonal pattern. The body surface area and volume of the nuclei of spermatids and spermatozoa in each maturation phase were analyzed using FIB/SEM tomography. The body surface area and volume of the nuclei decreased during spermatid maturation into spermatozoa. The RNE converged at the sperm neck and possessed a honeycomb structure. The method used revealed that the nuclei of spermatids gradually condense as they mature into spermatozoa. This method may be used to analyze small tissues, such as RNE, and detect morphological abnormalities in microtissues, such as spermatozoa.

Three-Dimensional Ultrastructural Analysis of the Head-Most Mitochondrial Roots of Mice Spermatozoa Using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Tomography.

This study aimed to clarify the three-dimensional ultrastructure of head-side mice spermatozoa mitochondria. Six 13-week-old male C57BL/6 mice were deeply anesthetized, perfused with 2% paraformaldehyde, and placed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for electron microscopy. After perfusion, the vas deferens was removed, and the specimens were cut into small cubes and subjected to postfixation and en bloc staining. Three-dimensional ultrastructural analysis was performed on five mitochondria on the spermatozoa head using conventional transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. Conventional TEM analysis showed that head-side mitochondria were not spiral in morphology but clearly horizontal to the sperm axis. However, this was difficult to evaluate further using conventional TEM. In the FIB/SEM analysis, the first and second head-most mitochondria were flat and straight, with no helix, and shaped as an attachment plug with two electrodes, and their tail side contacted the third mitochondrion. The third mitochondrion was shorter than the fourth and fifth and had a semicircular arching structure. The fourth and fifth mitochondria were spiral-shaped and intertwined. The redundant nuclear envelope encircled the head-most mitochondria. This ultrastructural analysis clarified that the head-most mitochondria have a unique morphology.

Mesoscopic structural analysis via deep learning processing, with a special reference to in vitro alteration in collagen fibre induced by a gap junction inhibitor.

Dense connective tissue, including the ligament, tendon, fascia and cornea, is formed by regularly arranged collagen fibres synthesized by fibroblasts (Fbs). The mechanism by which fibre orientation is determined remains unclear. Periodontal ligament Fbs consistently communicate with their surroundings via gap junctions (GJs), leading to the formation of a wide cellular network. A method to culture Fb-synthesized collagen fibres was previously reported by Schafer et al. ('Ascorbic acid deficiency in cultured human fibroblasts'. J. Cell Biol. 34: 83-95, 1967). This method has been applied to investigate the ability and activity of Fb collagen synthesis/phagocytosis using conventional electron microscopy (EM). However, the three-dimensional mesoscopic architecture of collagen fibres and the influence of GJ inhibitors on collagen fibre formation in vitro are poorly understood. In this study, three-dimensional mesoscopic analysis was used to elucidate the mechanism of directional fibre formation. We investigated the influence of GJ inhibitors on collagen formation driven by periodontal ligament Fbs in vitro, histomorphometrically, and the structural properties of in vitro collagen fibre on a mesoscale quantitatively, using correlative light and EM optimized for picrosirius red staining and focused ion beam-scanning EM tomography. Our results indicate that under culture conditions, in the presence of a GJ inhibitor, the orientation of collagen fibres becomes more disordered than that in the control group. This suggests that the GJ might be involved in determining fibre orientation during collagen fibre formation. Elucidation of this mechanism may help develop novel treatment strategies for connective tissue orientation disorders. Graphical Abstract.

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.

Correlative volume-imaging using combined array tomography and FIB-SEM tomography with beam deceleration for 3D architecture visualization in tissue.

Focused ion beamed (FIB) SEM has a higher spatial resolution than other volume-imaging methods owing to the use of ion beams. However, in this method, it is challenging to analyse entire biological structures buried deep in the resin block. We developed a novel volume-imaging method by combining array tomography and FIB-SEM tomography and investigated the chondrocyte ultrastructure. Our method imparts certainty in determining the analysis area such that cracks or areas with poor staining within the block are avoided. The chondrocyte surface showed fine dendritic processes that were thinner than ultrathin sections. Upon combination with immunostaining, this method holds promise for analysing mesoscopic architectures.

Three-dimensional ultrastructure and histomorphology of mouse circumvallate papillary taste buds before and after birth using focused ion beam-scanning electron microscope tomography.

Early taste buds are formed from placode cells. Placode cells differentiate into Type I-Ⅲ cells at birth; however, the ultrastructure of these first taste cells remain elusive. Here, we used focused ion beam-scanning electron microscopy (FIB-SEM) to analyze taste buds on the dorsal surface of the circumvallate papilla on embryonic day (E) 18.5 and postnatal day (P) 1.5. The taste buds on E18.5 existed as a mass of immature cells. One of the immature cells extended the cell process to the surface of the epithelium from the taste bud mass. Cytoplasm of this cell contained many mitochondria and vesicles in the apical region. The taste buds at P1.5 had small taste pores and had an onion-shaped structure. Most of the cells in the taste buds extended toward the taste pores. Some of the cells in the taste buds were Type II-like cells with glycogen in their cytoplasm. In this study, it was shown in three dimensions that immature cells extend to the surface of epithelium before the formation of the taste pore. Subsequently, the formation of taste pores and maturation of taste buds progress simultaneously.

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.

PDGFRα+ subepithelial interstitial cells act as a pacemaker to drive smooth muscle of the guinea pig seminal vesicle.

Smooth muscle cells (SMCs) of the guinea pig seminal vesicle (SV) develop spontaneous phasic contractions, Ca2+ flashes and electrical slow waves in a mucosa-dependent manner, and thus it was envisaged that pacemaker cells reside in the mucosa. Here, we aimed to identify the pacemaker cells in SV mucosa using intracellular microelectrode and fluorescence Ca2+ imaging techniques. Morphological characteristics of the mucosal pacemaker cells were also investigated using focused ion beam/scanning electron microscopy tomography and fluorescence immunohistochemistry. Two populations of mucosal cells developed spontaneous Ca2+ transients and electrical activity, namely basal epithelial cells (BECs) and subepithelial interstitial cells (SICs). Pancytokeratin-immunoreactive BECs were located on the apical side of the basement membrane (BM) and generated asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). The spontaneous Ca2+ transients and STDs were not diminished by 10 µM nifedipine but abolished by 10 µM cyclopiazonic acid (CPA). Platelet-derived growth factor receptor α (PDGFRα)-immunoreactive SICs were distributed just beneath the basal side of the BM and developed synchronous Ca2+ oscillations and electrical slow waves, which were suppressed by 3 µM nifedipine and abolished by 10 µM CPA. In SV mucosal preparations in which some smooth muscle bundles remained attached, SICs and residual SMCs developed temporally correlated spontaneous Ca2+ transients. Neurobiotin injected into SICs spread not only to neighbouring SICs but also to neighbouring SMCs or vice versa. These results suggest that PDGFRα+ SICs electrotonically drive the spontaneous contractions of SV smooth muscle. KEY POINTS: In many visceral smooth muscle organs, spontaneous contractions are electrically driven by non-muscular pacemaker cells. In guinea pig seminal vesicles (SVs), as yet unidentified mucosal cells appear to drive neighbouring smooth muscle cells (SMCs). Two populations of spontaneously active cells are distributed in the SV mucosa. Basal epithelial cells (BECs) generate asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). In contrast, subepithelial interstitial cells (SICs) develop synchronous Ca2+ oscillations and electrical slow waves. Pancytokeratin-immunoreactive (IR) BECs are located on the apical side of the basement membrane (BM), while platelet-derived growth factor receptor α (PDGFRα)-IR SICs are located on the basal side of the BM. Spontaneous Ca2+ transients in SICs are synchronised with those in SV SMCs. Dye-coupling between SICs and SMCs suggests that SICs act as pacemaker cells to drive the spontaneous contractions of SV smooth muscle.

3D mesoscopic architecture of a heterogeneous cellular network in the cementum-periodontal ligament-alveolar bone complex.

Cell-to-cell communication orchestrates various cell and tissue functions. This communication enables cells to form cellular networks with each other through direct contact via intercellular junctions. Because these cellular networks are closely related to tissue and organ functions, elucidating the morphological characteristics of cellular networks could lead to the development of novel therapeutic approaches. The tooth, periodontal ligament (PDL) and alveolar bone form a complex via collagen fibres. Teeth depend on the co-ordinated activity of this complex to maintain their function, with cellular networks in each of its three components. Imaging methods for three-dimensional (3D) mesoscopic architectural analysis include focused ion beam/scanning electron microscopy (FIB/SEM), which is characterized by its ability to select observation points and acquire data from complex tissue after extensive block-face imaging, without the need to prepare numerous ultrathin sections. Previously, we employed FIB/SEM to analyse the 3D mesoscopic architecture of hard tissue including the PDL, which exists between the bone and tooth root. The imaging results showed that the cementum, PDL and alveolar bone networks are in contact and form a heterogeneous cellular network. This cellular network may orchestrate mechanical loading-induced remodelling of the cementum-PDL-alveolar bone complex as the remodelling of each complex component is coordinated, as exemplified by tooth movement due to orthodontic treatment and tooth dislocation due to occlusal loss. In this review, we summarize and discuss the 3D mesoscopic architecture of cellular networks in the cementum, PDL and alveolar bone as observed in our recent mesoscopic and morphological studies.

Distribution, shape, and immunohistochemical characteristics of serotonin-immunoreactive neuroendocrine cells in the urethra and periurethral genital organs in mice.

The aim of this study is to clarify the disibution, shape, and immunohistochemical characteristics of serotonin-immunoreactive neuroendocrine cells (SIR-NECs) in mouse prostate and in the surrounding genital organs by histological and immunohistochemical analysis of the light microscopic serial sections of urethra. We collected lower urinary tracts from 13-week-old mice and observed the distribution pattern and shape of the SIR-NECs by serial light microscopy. The organs on the sections were divided into three anatomical zones to clarify the distribution pattern of SIR-NECs: (1) zone A, the ducts near the prostatic urethra; (2) zone B, the ducts outside the urethral sphincter; and (3) zone C, the acinus areas. Sections were double immune-stained with antibodies against serotonin and one of neuroendocrine-related factors (NRFs), including 10 neural cell markers and eight neurotransmitters, and also 4',6-diamino-2-phenylindole (DAPI). In addition, SIR-NECs were double immune-stained with antibodies against cytokeratin 5 (CK5) and p63, together with DAPI. SIR-NECs were mostly localized in zone A, and no SIR-NECs were observed in zone C. The proportion of flask-shaped SIR-NECs was approximately 15% in zones A and B. No flask-shaped SIR-NECs were observed in urethral epithelia. The NRFs co-localized with SIR-NEC were calcitonin gene-related peptide, CD56, chromogranin A, neuron-specific enolase, neuron cytoplastic protein 9.5, and synaptophysin (72.3%, 73.2%, 88.9%, 92.3%, 91.7%, and 81.9%, respectively). CK5 and p63 were not co-localized with SIR-NECs.　In this study, SIR-NEC of the urethra and the surrounding genital organs was ubiquitous in the urethra and the ducts near the urethra and co-expressed specific nerve-related NRFs.

Comparison of Structural Properties Between Postnatal and Adult Tendon Insertion with FIB/SEM Tomography in Rat.

OBJECTIVE: The repaired tendon-bone interface after rotator cuff (RC) repair has been identified as a mechanical weak point, which may contribute to re-tearing. Analyzing the postnatal development of a normal tendon insertion in detail may be useful in helping to promote the regeneration of a normal tendon insertion. We verified the morphological differences between postnatal and adult tendon insertions in terms of the cellular structural properties using FIB/SEM tomography. MATERIALS AND METHOD: SPostnatal and adult Sprague-Dawley rats were used as a model of tendon insertion. The morphological structure of the insertion was evaluated using hematoxylin and eosin (HE) staining, and the 3D ultrastructure of the cells in the insertion was evaluated using FIB/SEM tomography. Additionally, the volume of the cell bodies, nuclei, and cytoplasm were measured and compared in a quantitative analysis. RESULTS: On conventional histology, the boundary line between the fibrocartilage and mineralized cartilage was flat in the adult insertions; however, the boundary line between the mineralized cartilage and bone formed deep interdigitations. The morphology of the cells among the collagen bundles in the adult insertions was completely different from those in the postnatal insertions at the 3D ultrastructural level. The cellular structural properties were statistically different between the postnatal and adult insertions. CONCLUSIONS: In the present study, the morphological differences between postnatal and adult tendon insertion in terms of the ultrastructural cellular properties were clarified. These findings may aid in determining how to regenerate a clinically stable tendon insertion at the tendon-bone interface after RC repair.

Morphological analysis of interstitial cells in murine epididymis using light microscopy and transmission electron microscopy.

Smooth muscle contraction of the epididymis plays an important role in sperm transport. Although PDGFRα-positive interstitial cells (PDGFRα (+) ICs) are thought to be involved in controlling smooth muscle movement via intercellular signaling, they have not yet been reported to date in the epididymis. Therefore, we aimed to investigate the morphological characteristics of PDGFRα (+) ICs in the interstitial space of the murine epididymis. Immunohistochemistry showed that PDGFRα (+) ICs co-labeled with CD34 (PDGFRα (+) CD34 (+) ICs were distributed in the interstitial space of the murine epididymis from the initial segment (IS) to the cauda of the epididymis. PDGFRα (+) ICs that were not co-labeled with CD34 (PDGFRα (+) CD34 (-) ICs) were observed just beneath the epithelium from the corpus to the cauda but not in the IS. Both types of PDGFRα (+) ICs were in close proximity to each other as well as the surrounding nerves and macrophages. In addition, PDGFRα (+) CD34 (-) ICs beneath the epithelium were also in close proximity to the basal cells. Using transmission electron microscopy, we identified ICs that possessed elongated and woven cellular processes and were in close proximity to each other, surrounding the cells in the interstitial space. In the murine epididymis, it is suggested that there are two subtypes of ICs that show different distribution patterns depending on the segment, which may reflect segmental differences in mechanisms of sperm transport, forming a cellular network by physical interactions in the murine epididymis.

Identification of PDGFRα-positive interstitial cells in the distal segment of the murine vas deferens.

Platelet-derived growth factor receptor-α (PDGFRα)-positive interstitial cells (ICs) are widely distributed in various organs and may be involved in the motility of various tubular organs. We, for the first time, aimed to investigate the distribution, immunohistochemical characteristics, and ultrastructure of PDGFRα-positive ICs in murine vas deferens, using confocal laser scanning microscopy, transmission electron microscopy (TEM), and immuno-electron microscopy (immuno-EM). For immunofluorescence, we used antibodies against PDGFRα and other markers of ICs. PDGFRα-positive ICs were distributed widely in the lamina propria, smooth muscles, and serosal layers. Although most PDGFRα-positive ICs labeled CD34, they did not label CD34 in the subepithelial layers. Additionally, PDGFRα-positive ICs were in close proximity to each other, as also to the surrounding cells. TEM and immuno-EM findings revealed that PDGFRα-positive ICs established close physical interactions with adjacent ICs. Extracellular vesicles were also detected around the PDGFRα-positive ICs. Our morphological findings suggest that PDGFRα-positive ICs may have several subpopulations, which can play an important role in intercellular signaling via direct contact with the IC network and the extracellular vesicles in the murine vas deferens. Further investigation on PDGFRα-positive ICs in the vas deferens may lead to understanding the vas deferens mortility.

A simple preparation method for CLEM using pre-embedding immunohistochemistry with a novel fluorescent probe and stable embedding resin.

Correlative light and electron microscopy (CLEM) is an excellent approach for examining the cellular localization of biomolecules. Here, we developed a simple method for CLEM by combining pre-embedding immunohistochemistry with a novel fluorescent probe, namely Fluolid NS Orange, and an embedding resin called 'Durcupan™'. Specimens were embedded in Durcupan™ or LR White after immunolabeling and post-fixation using glutaraldehyde and osmium tetroxide. Next, ultrathin sections were prepared on a finder grid with navigation markers. The section of the specimen embedded in Durcupan™ was found to be more stable against electron beam irradiation than specimens embedded in LR White. A fluorescence light microscopy image and a transmission electron microscopy (TEM) image, at wide-field, and low magnification, were independently obtained with the same ultrathin section. Using the three corners between finder grid bars as landmarks, fluorescence light microscopy images were superimposed with wide-field, low-magnification TEM images to identify the region of interest, which was subsequently enlarged to ascertain cellular structures localized beneath fluorescent signals. However, the enlarged TEM images appeared blurred, and fluorescence signals had a hazy appearance. To resolve this, the enlarged TEM images were replaced by high-resolution TEM images focused directly on the region of interest, thereby facilitating the collection of high-resolution CLEM images. The simple sample processing method for CLEM using osmium-resistant Fluolid NS Orange and electron beam damage-resistant Durcupan™ allowed the determination of the precise localization of fluorescence signals at subcellular levels.

Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM.

Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

Sex Differences in Dendritic Spine Formation in the Hippocampus and Animal Behaviors in a Mouse Model of Hyperthyroidism.

Thyroid hormones are critical for the regulation of development and differentiation of neurons and glial cells in the central nervous system (CNS). We have previously reported the sex-dependent changes of glial morphology in the brain under the state of hyperthyroidism. Here, we examined sex-dependent changes in spine structure of granule neurons in the dentate gyrus of hippocampus in male and female mice with hyperthyroidism. Using FIB/SEM (focused ion beam/scanning electron microscopy), three-dimensional reconstructed structures of dendritic spines in dentate granule cells were analyzed. Dendritic spine density in granule cells increased significantly in both male and female mice with hyperthyroidism. The decrease in spine volume was observed only in female mice. These findings suggest that hyperthyroidism induces the formation of spines with normal size in male mice but the formation of spines with small size in female mice. To evaluate an outcome of neuronal and previously observed glial changes, behavioral tests were performed. Male mice with hyperthyroidism showed increased locomotor activity in the open field test, while female mice showed elevated immobility time in the tail suspension test, reflecting depression-like behavior. Although direct link between changes in spine and behavioral modifications requires further analysis, our results may help to understand gender-dependent neurological and psychological symptoms observed in patients with hyperthyroidism.