Size and Surface Properties of Functionalized Organosilica Particles Impact Cell-Particle Interactions Including Mitochondrial Activity.

Understanding of the interactions between macrophages and multifunctional nanoparticles is important for development of novel macrophage-based immunotherapies. Here, we investigated the effects of fluorescent thiol-organosilica particle size and surface properties on cell-particle interactions, including mitochondrial activity, using the mouse macrophage cell line J774A.1. Three different sizes of thiol-organosilica particles (150, 400, and 680 nm in diameter) containing fluorescein (OS/F150, OS/F400, and OS/F680) and particles surface functionalized with polyethylenimine (PEI) (OS/F150PEI, OS/F400PEI, and OS/F680PEI) were prepared. Flow cytometric analysis, time-lapse imaging, and single-cell analysis of particle uptake and mitochondrial activity of J774A.1 cells demonstrated variations in uptake and kinetics depending on the particle size and surface as well as on each individual cell. Cells treated with OS/F150 and OS/F150PEI showed higher uptake and mitochondrial activity than those treated with other particles. The interaction between endosomes and mitochondria was observed using 3D fluorescent imaging and was characterized by the involvement of iron transport into mitochondria by iron-containing proteins adsorbed on the particle surface. Scanning electron microscopy of the cells treated with the particles revealed alterations in cell membrane morphology, depending on particle size and surface. We performed correlative light and electron microscopy combined with time-lapse and 3D imaging to develop an integrated correlation analysis of particle uptake, mitochondrial activity, and cell membrane morphology in single macrophages. These cell-specific characteristics of macrophages against functional particles and their evaluation methods are crucial for understanding the immunological functions of individual macrophages and developing novel immunotherapies.

Histological study on the reginal difference in the localization of mucosal enteric glial cells and their sheath structure in the rat intestine.

The present study aimed to histologically clarify the regional specificity of the mucosal enteric glial cells (mEGCs) in the rat intestine with serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM analysis with the ileum, the cecum and the descending colon revealed that mEGC nuclei were more abundant in the data stacks from the apical portion of the villus and the lateral portion of the crypt of the ileum. mEGCs exhibited a high rate of coverage over the nerve bundle around the lateral portion of the ileal crypt, but showed an extremely low level of coverage in the luminal portion of the cecum. These findings evidenced regional differences in the localization of mEGCs and in their sheath structure in the rat intestine.

Analysis of cell-nanoparticle interactions and imaging of in vitro labeled cells showing barcorded endosomes using fluorescent thiol-organosilica nanoparticles surface-functionalized with polyethyleneimine.

Biomedical imaging using cell labeling is an important technique to visualize cell dynamics in the body. To label cells, thiol-organosilica nanoparticles (thiol-OS) containing fluorescein (thiol-OS/Flu) and rhodamine B (thiol-OS/Rho) were surface-functionalized with polyethyleneimine (PEI) (OS/Flu-PEI and OS/Rho-PEI) with 4 molecular weights (MWs). We hypothesized PEI structures such as brush, bent brush, bent lie-down, and coiled types on the surface depending on MWs based on dynamic light scattering and thermal gravimetric analyses. The labeling efficacy of OS/Flu-PEIs was dependent on the PEI MW and the cell type. A dual-particle administration study using thiol-OS and OS-PEIs revealed differential endosomal sorting of the particles depending on the surface of the NPs. The endosomes in the labeled cells using OS/Flu-PEI and thiol-OS/Rho revealed various patterns of fluorescence termed barcoded endosomes. The cells labeled with OS-PEI in vitro were administrated to mice intraperitoneally after in situ labeling of peritoneal cells using thiol-OS/Rho. The in vitro labeled cells were detected and identified in cell aggregates in vivo seamlessly. The labeled cells with barcoded endosomes were also identified in cell aggregates. Biomedical imaging of in vitro OS-PEI-labeled cells combined with in situ labeled cells showed high potential for observation of cell dynamics.

Differences in interaction lead to the formation of different types of insulin amyloid.

Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.

Correlative light and electron microscopy of poly(ʟ-lactic acid) spherulites for fast morphological measurements using a convolutional neural network.

Polarized optical microscopy (POM) and transmission electron microscopy (TEM) are widely used for imaging polymer spherulite structures. TEM provides nanometer resolution to image small spherulites of sub-micrometer in diameter, while POM is more suitable for observing large spherulites. However, high-resolution images with a large field of view are challenging to achieve due to the deformations of ultrathin sectioned samples used for TEM observations. In this study, we demonstrated that correlative light and electron microscopy (CLEM) combining POM and TEM could effectively characterize the spherulite structures in a wide range from nanometer to several hundred micrometers that neither TEM nor POM alone could cover. Furthermore, the deformations of the TEM ultrathin sections were corrected by referencing to the POM images at the same position of the sample, and large-area TEM images without deformations were successfully produced. The spherulite structures of poly(ʟ-lactic acid) were successfully analyzed using CLEM and TEM in a wide range of spatial scales at the same field of view. The large-area TEM image (250 µm × 250 µm), consisting of 702 TEM images stitched together, was subjected to machine learning to extract the essential structural information of spherulites. Analysis using the convolutional neural network, a well-known algorithm You Only Look Once (YOLO), demonstrated that it was practical to accurately obtain the diameter distribution and the space-filling factor (relative crystallinity) of the spherulites. This study presents a new approach for acquiring high-resolution images with a large field of view and processing the images at a fast speed.

Ultrastructural and phenotypical diversity of macrophages in the rat ileal mucosa.

Several types of macrophages have been reported in the intestinal mucosa, but their histological localization remains ambiguous. Here, we obtained detailed information about ultrastructural and phenotypical diversity of macrophage-like cells (MLCs) in the rat ileal mucosa using immunofluorescent analysis and serial block-face scanning electron microscopy (SBF-SEM). The results revealed that the cells immunopositive for CD68, the pan-macrophage marker, included CD163-CD4+, CD163+CD4+, and CD163-CD4- cells in the lamina propria (LP) of the intestinal villus and around the crypt. CD68+CD4+CD163- cells seemed to be preferentially localized in the intestinal villus, whereas CD68+CD163+CD4+ cells were frequently localized around the crypt. SBF-SEM analysis identified three types of MLCs in the ileal mucosa, which were tentatively named types I-III MLC based on aspects of the 3D-ultrastructure, such as the localization, quantity of lysosomes, endoplasmic reticulum, and exoplasm. Type I and II MLCs were localized in the villous LP, while type III MLCs were localized around the crypt, although type II MLCs were a minor population. All three MLC types extended their cellular processes into the epithelium, with type I MLCs showing the greatest abundance of extended processes. Type I MLCs in the upper portion of the intestinal villus showed a higher level of attachment to intraepithelial lymphocytes (IELs) compared to type III MLCs around the crypt. These findings suggest that macrophages of the rat ileal mucosa differed by region along the longitudinal axis of the villous tip-crypt from the perspective of ultrastructure, cellular composition, localization, and interactions with IELs.

Membranous Structures Directly Come in Contact With p62/SQSTM1 Bodies.

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.

Three-dimensional analysis of neural connectivity with cells in rat ileal mucosa by serial block-face scanning electron microscopy.

The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we investigated the comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs), macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat ileal mucosa.

Morphological and phenotypical diversity of eosinophils in the rat ileum.

Eosinophils are abundantly present in intestinal mucosa. However, the morphological characteristics of their cellular population are still largely unknown. In this study, we examine their characteristics in the rat ileal mucosa using histological and ultrastructural methods. The results indicated that ileal eosinophils could be distinguished into two main groups based on their nuclear shapes and distribution: eosinophils with spheric or reniform nuclei mainly localized in the villous region and eosinophils with annular or bacilliform nuclei as the major population around crypts. Immunohistochemical analysis revealed that all eosinophils in the lamina propria (LP) were immunopositive for CD11b, whereas eosinophils in LP of the intestinal villus but not those in LP around the crypt, were immunopositive for CD11c. Three-dimensional ultrastructural analysis using serial block-face scanning electron microscopy showed that the eosinophils with spheric or reniform nuclei were abundant in the upper portions of the intestinal villus, whereas those with annular nuclei were abundant in the lower portions of the intestinal villus and around crypts. The eosinophils with spheric or reniform nuclei possessed broader cellular bodies with greater abundance of surface projections compared with those with annular nuclei. Eosinophils in the upper portions of intestinal villus frequently extended their cellular bodies into the intraepithelial space. The number of total and eosinophil-specific granules was positively correlated with the minor axis of the nuclear holes in the annular nuclei. These data suggest that ileal eosinophils exhibit not homogenous but rather diverse characteristics, possible due to the mixture of eosinophils at different maturation and/or activation stages.

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.

Three-dimensional analysis of fibroblast-like cells in the lamina propria of the rat ileum using serial block-face scanning electron microscopy.

Serial block-face scanning electron microscopy (SBF-SEM) is useful for three-dimensional observation of tissues or cells at high-resolution. In this study, SBF-SEM was used to three-dimensionally analyze the characteristics of fibroblast-like cells (FBLCs) in the rat ileal lamina propria (LP). The results revealed that FBLCs in LP could be divided into four types, tentatively named FBLC type I-IV, based on the external cellular appearance, abundance or shape of each organelle, detailed distribution in the LP and relationship with surrounding cells. FBLC-I and -II localized around the intestinal crypt (InC), FBLC-III localized from the lateral portion of InC to the apical portion of the intestinal villus (InV), and FBLC-IV localized in the InV. FBLC-I, -II and -III, but not FBLC-IV, localized beneath the epithelium. FBLC-II possessed thin lamellar-shaped endoplasmic reticula, whereas FBLC-I possessed expanded endoplasmic reticula that occasionally showed a spherical shape. FBLC-III and -IV possessed a cytoplasmic region with high-electron density and no organelles immediately beneath the cellular membrane; this region was found at the epithelial sides in FBLC-III and scattered in FBLC-IV. FBLC-IV were in constant close proximity to villous myocytes throughout the InV and also in contact with FBLC-III especially in the apical portion of the InV. FBLC-I, -II and -IV, but not -III, were constantly found to be in contact with various immunocompetent cells in the LP. Three-dimensional analysis using SBF-SEM indicates that four types of FBLC localized in the rat ileal LP.

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.

Fabrication and characterization of sample-supporting film made of silicon nitride for large-area observation in transmission electron microscopy.

In transmission electron microscopy (TEM), silicon nitride (SiN) films are widely used as sample-supporting films owing to their robustness. We fabricated large-scale SiN films deposited by low-pressure chemical vapor deposition (LPCVD). This preparation method is advantageous for large window areas, since it yields films with control over properties such as tension and thickness. We fabricated large SiN windows for mounting large ultrathin sections and for acquiring large-area TEM images. Thus, sample sections sliced by conventional sample preparation techniques were successfully mounted on these sample-supporting films. We successfully obtained a 680 × 250 µm2 TEM montage image of a whole Drosophila embryo.

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.

Characterization of paramyosin and thin filaments in the smooth muscle of acorn worm, a member of hemichordates.

Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca2+-dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca2+-independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles.

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies.

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

The proximal half of the Drosophila E-cadherin extracellular region is dispensable for many cadherin-dependent events but required for ventral furrow formation.

The formation of the ventral furrow during Drosophila gastrulation is driven by coordinated apical constriction. Cell-cell adhesion is thought to regulate apical constriction, but the mechanisms are poorly understood. DE-cadherin, an epithelial classic cadherin, has in its membrane-proximal extracellular region a suite of domains absent from vertebrate/urochordate classic cadherins. We constructed DEΔP, a DE-cadherin derivative that lacks the membrane-proximal half of the extracellular region but retains the entire cytoplasmic domain and still exhibits strong cell-cell binding ability. The extracellular region of DEΔP consists of only cadherin repeats, mimicking vertebrate/urochordate classic cadherins. In animals lacking DE-cadherin, DEΔP organized adherens junction assembly and functioned fully in many cadherin-dependent processes, including oogenesis. Embryos in which DE-cadherin was entirely replaced by DEΔP established the blastoderm epithelium but failed to form a ventral furrow. Apical constrictions were initiated relatively normally but subsequently decelerated. These were then followed by catastrophic disruption of the junctional network. Our results suggest that although the membrane-proximal half of the DE-cadherin extracellular region is dispensable for many developmental events, it is essential for efficient and robust apical constriction during ventral furrow formation.