Development of a novel zebrafish model of diabetic nephropathy.

Diabetic nephropathy (DN) is a substantial healthcare challenge as a complication of diabetes, owing to the high risk of morbidity and mortality involved. Although significant progress has been made in understanding the pathogenesis of DN, more efficient models are required to develop new therapeutics. Here, we created a DN model in zebrafish by crossing diabetic Tg(acta1:dnIGF1R-EGFP) and proteinuria-tracing Tg(l-fabp::VDBP-GFP) lines, named zMIR/VDBP. Overfed adult zMIR/VDBP fish developed severe hyperglycemia and proteinuria, which were not observed in wild-type zebrafish. Renal histopathology revealed human DN-like characteristics, such as glomerular basement membrane thickening, foot process effacement, and glomerular sclerosis. Glomerular dysfunction is restored upon calorie restriction. RNA sequencing (RNA-seq) analysis demonstrated that zebrafish DN kidneys exhibited transcriptional patterns similar to human DN pathogenesis. Notably, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was activated, a phenomenon observed in the early phase of human DN. In addition, metformin improved hyperglycemia and proteinuria in DN zebrafish by modulating AKT phosphorylation. Our results indicated that zMIR/VDBP fish are suitable for elucidating the mechanisms underlying human DN and could be a powerful tool for therapeutic discovery.

Treatment of tubular damage in high-fat-diet-fed obese mice using sodium-glucose co-transporter inhibitors.

A long-term high-fat diet (HFD) causes obesity and changes in renal lipid metabolism and lysosomal dysfunction in mice, causing renal damage. Sodium-glucose co-transporter inhibitors, including phlorizin, exert nephroprotective effects in patients with chronic kidney disease, but the underlying mechanism remains unclear. A HFD or standard diet was fed to adult C57BL/6J male mice, and phlorizin was administered. Lamellar body components of the proximal tubular epithelial cells (PTECs) were investigated. After phlorizin administration in HFD-fed mice, sphingomyelin and ceramide in urine and tissues were assessed and label-free quantitative proteomics was performed using kidney tissue samples. Mitochondrial elongation by fusion was effective in the PTECs of HFD-fed obese mice under phlorizin administration, and many lamellar bodies were found in the apical portion of the S2 segment of the proximal tubule. Phlorizin functioned as a diuretic, releasing lamellar bodies from the apical membrane of PTECs and clearing the obstruction in nephrons. The main component of the lamellar bodies was sphingomyelin. On the first day of phlorizin administration in HFD-fed obese mice, the diuretic effect was increased, and more sphingomyelin was excreted through urine than in vehicle-treated mice. The expressions of three peroxisomal ß-oxidation proteins involved in fatty acid metabolism were downregulated after phlorizin administration in the kidneys of HFD-fed mice. Fatty acid elongation protein levels increased with phlorizin administration, indicating an increase in long-chain fatty acids. Lamellar bodies accumulated in the proximal renal tubule of the S2 segment of the HFD-fed mice, indicating that the urinary excretion of lamellar bodies has nephroprotective effects.

Ultrastructural characteristics of oligodendrocyte precursor cells in the early postnatal mouse optic nerve observed by serial block-face scanning electron microscopy.

Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs.

CTCF loss induces giant lamellar bodies in Purkinje cell dendrites.

CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as "giant lamellar bodies" (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.

Astrocytic dysfunction induced by ABCA1 deficiency causes optic neuropathy.

Astrocyte abnormalities have received great attention for their association with various diseases in the brain but not so much in the eye. Recent independent genome-wide association studies of glaucoma, optic neuropathy characterized by retinal ganglion cell (RGC) degeneration, and vision loss found that single-nucleotide polymorphisms near the ABCA1 locus were common risk factors. Here, we show that Abca1 loss in retinal astrocytes causes glaucoma-like optic neuropathy in aged mice. ABCA1 was highly expressed in retinal astrocytes in mice. Thus, we generated macroglia-specific Abca1-deficient mice (Glia-KO) and found that aged Glia-KO mice had RGC degeneration and ocular dysfunction without affected intraocular pressure, a conventional risk factor for glaucoma. Single-cell RNA sequencing revealed that Abca1 deficiency in aged Glia-KO mice caused astrocyte-triggered inflammation and increased the susceptibility of certain RGC clusters to excitotoxicity. Together, astrocytes play a pivotal role in eye diseases, and loss of ABCA1 in astrocytes causes glaucoma-like neuropathy.

Ddx20, DEAD box helicase 20, is essential for the differentiation of oligodendrocyte and maintenance of myelin gene expression.

Oligodendrocytes form myelin sheaths that surround axons, contributing to saltatory conduction and proper central nervous system (CNS) function. Oligodendrocyte progenitor cells (OPCs) are generated during the embryonic stage and differentiate into myelinating oligodendrocytes postnatally. Ddx20 is a multifunctional, DEAD-box helicase involved in multiple cellular processes, including transcription, splicing, microRNA biogenesis, and translation. Although defects in each of these processes result in abnormal oligodendrocyte differentiation and myelination, the involvement of Ddx20 in oligodendrocyte terminal differentiation remains unknown. To address this question, we used Mbp-Cre mice to generate Ddx20 conditional knockout (cKO) mice to allow for the deletion of Ddx20 from mature oligodendrocytes. Mbp-Cre;Ddx20 cKO mice demonstrated small body sizes, behavioral abnormalities, muscle weakness, and short lifespans, with mortality by the age of 2 months old. Histological analyses demonstrated significant reductions in the number of mature oligodendrocytes and drastic reductions in the expression levels of myelin-associated mRNAs, such as Mbp and Plp at postnatal day 42. The number of OPCs did not change. A thin myelin layer was observed for large-diameter axons in Ddx20 cKO mice, based on electron microscopic analysis. A bromodeoxyuridine (BrdU) labeling experiment demonstrated that terminal differentiation was perturbed from ages 2 weeks to 7 weeks in the CNS of Mbp-Cre;Ddx20 cKO mice. The activation of mitogen-activated protein (MAP) kinase, which promotes myelination, was downregulated in the Ddx20 cKO mice based on immunohistochemical detection. These results indicate that Ddx20 is an essential factor for terminal differentiation of oligodendrocytes and maintenance of myelin gene expression.

Large-scale electron microscopic volume imaging of interfascicular oligodendrocytes in the mouse corpus callosum.

Single oligodendrocytes produce myelin sheaths around multiple axons in the central nervous system. Interfascicular oligodendrocytes (IOs) facilitate nerve conduction, but their detailed morphologies remain largely unknown. In the present study, we three-dimensionally reconstructed IOs in the corpus callosum of adult mouse using serial block face scanning electron microscopy. The cell bodies of IOs were morphologically polarized and extended thick processes from the cytoplasm-rich part of the cell. Processes originating from the cell body of each IO can be classified into two types: one myelinates an axon without branching, while the other type branches and each branch myelinates a distinct axon. Myelin sheaths originating from a particular IO have biased thicknesses, wrapping axons of a limited range of diameters. Consistent with this finding, IOs transduced and visualized with a rabies viral vector expressing GFP showed statistically significant variation in their myelination patterns. We further reconstructed the sheath immediately adjacent to that derived from each of the analyzed IOs; the thicknesses of the pair of sheaths were significantly correlated despite emanating from different IOs. These results suggest that a single axon could regulate myelin sheath thicknesses, even if the sheaths are derived from distinct IOs. Collectively, our results indicate that the IOs have their own myelin profiles defined by myelin thickness and axonal diameter although axons may regulate thickness of myelin sheath.

Radiometric Calibration for a Multispectral Sensor Onboard RISESAT Microsatellite Based on Lunar Observations.

Radiometric calibration utilizing the Moon as a reference source is termed as lunar calibration. It is a useful method for evaluating the performance of optical sensors onboard satellites orbiting the Earth. Lunar calibration provides sufficient radiometric calibration opportunities without requiring any special equipment, and is suitable for nano/microsatellites. This study applies lunar calibration to a multispectral sensor, Ocean Observation Camera (OOC), on board a microsatellite named Rapid International Scientific Experiment Satellite. Simulating the brightness of the Moon based on the RObotic Lunar Observatory and SELENE/Spectrum Profiler models, sensitivity degradation was proven to be negligible in any of the four spectral bands of the OOC with the sensor temperature correction. A bluing trend in the OOC's sensor sensitivity was revealed, indicating a shorter observation wavelength shows larger irradiance. Comparing the top-of-atmosphere reflectance of Railroad Valley Playa with the Radiometric Calibration Network dataset revealed that the derived calibration parameter from the lunar calibration was valid for correcting the bluing trend in the visible range. Although the lunar and vicarious calibration parameters for the infrared band were unexpectedly inconsistent, lunar calibration could potentially contribute toward estimating the contaminated background radiance in the Earth observation images.

Marine biodiversity refugia in a climate-sensitive subarctic shelf.

The subarctic shelf of the Eastern Bering Sea (EBS) is one of the world's most productive marine environments, exposed to drastic climate changes characterized by extreme fluctuations in temperature, sea ice concentration, timing, and duration. These climatic changes elicit profound responses in species distribution, abundance, and community composition. Here, we examined the patterns of alpha and temporal beta diversity of 159 marine taxa (66 vertebrates and 93 invertebrate species) from 29 years (1990-2018) of species observations from the NOAA bottom trawl surveys in the EBS. Based on these data, we identified geographically distinct refugial zones in the northern and southern regions of the middle shelf, defined by high species richness and similarity in community species composition over time. These refugial zones harbor higher frequencies of occurrence for representative taxa relative to the regions outside of refugia. We also explored the primary environmental factors structuring marine biodiversity distributions, which underpinned the importance of the winter sea ice concentration to alpha and temporal beta diversity. The spatial biodiversity distributions between high and low winter sea ice regimes highlighted contrasting signals. In particular, the latter showed elevated species richness compared to the former. Further, the temporal beta diversity between the high and low winter sea ice periods underpinned an overall increase in the compositional similarity of marine communities in the EBS. Despite these spatiotemporal differences in biodiversity distributions, the identified refugia represent safe havens of marine biodiversity in the EBS. Distinguishing these areas can help facilitate conservation and management efforts under accelerated and ongoing climatic changes.

Identification of skipjack tuna (Katsuwonus pelamis) pelagic hotspots applying a satellite remote sensing-driven analysis of ecological niche factors: A short-term run.

Skipjack tuna (SJT) pelagic hotspots in the western North Pacific (WNP) were modelled using fishery and satellite remotely sensed data with Ecological Niche Factor Analysis (ENFA) models. Our objectives were to model and predict habitat hotspots for SJT and assess the monthly changes in sub-surface temperatures and mixed layer depths at fishing locations. SJT presence-only monthly resolved data, sea surface temperature, chlorophyll-a, diffuse attenuation coefficient, sea surface heights and surface wind speed were used to construct ENFA models and generate habitat suitability indices using a short-term dataset from March-November 2004. The suitability indices were then predicted for July-October (2007 and 2008). Monthly aggregated polygons of areas fished by skipjack tuna pole and line vessels were also overlaid on the predicted habitat suitability maps. Distributions of sub-surface temperatures and mixed layer depths (MLD) at fishing locations were also examined. Our results showed good fit for ENFA models, as indicated by the absolute validation index, the contrast validation index and the continuous Boyce index. The predicted hotspots showed varying concurrences when compared with 25-degree polygons derived from fished areas. Northward shifts in SJT hotspots corresponded with declining MLDs from March to September. The MLDs were shallower in summer and deeper in autumn and winter months. The habitat hotspots modeled using ENFA were consistent with the known ecology and seasonal migration pattern of SJT. The findings of this work, derived from a short-term dataset, enable identification of SJT hotspots in the WNP, thus contributing valuable information for future research on SJT habitat prediction models.

Multiple facets of marine biodiversity in the Pacific Arctic under future climate.

Climate change is triggering a global reorganization of marine life. Biogeographical transition zones, diversity-rich regions straddling biogeographical units where many species live at, or close to, their physiological tolerance limits (i.e., range distribution edges), are redistribution hotspots that offer a unique opportunity to understand the mechanisms and consequences of climate-driven thermophilization processes in natural communities. In this context, we examined the impacts of climate change projections in the 21st century (2026-2100) on marine biodiversity in the Eastern Bering and Chukchi seas within the Pacific Arctic, a climatically exposed and sensitive boreal-to-Arctic transition zone. Overall, projected changes in species distributions, modeled using species distribution models, resulted in poleward increases in species richness and functional redundancy, along with pronounced reductions in phylogenetic distances by century's end (2076-2100). Future poleward shifts of boreal species in response to warming and sea ice changes are projected to alter the taxonomic and functional biogeography of contemporary Arctic communities as larger, longer-lived and more predatory taxa expand their leading distributional margins. Drawing from the existing evidence from other Arctic regions, these changes are anticipated to increase the susceptibility and vulnerability of the Arctic ecosystems, as trophic connectance between biological components increases, thus decreasing the modularity of Arctic food webs. Our results demonstrate how integrating multiple diversity facets can provide key insights into the relationships between climate change, species composition and ecosystem functioning across marine biogeographic regions.

Sodium-glucose co-transporter (SGLT) inhibitor restores lost axonal varicosities of the myenteric plexus in a mouse model of high-fat diet-induced obesity.

Diabetes impairs enteric nervous system functions; however, ultrastructural changes underlying the pathophysiology of the myenteric plexus and the effects of sodium-glucose co-transporter (SGLT) inhibitors are poorly understood. This study aimed to investigate three-dimensional ultrastructural changes in axonal varicosities in the myenteric plexus and the effect thereon of the SGLT inhibitor phlorizin in mice fed a high-fat diet (HFD). Three-dimensional ultrastructural analysis using serial block-face imaging revealed that non-treated HFD-fed mice had fewer axonal varicosities and synaptic vesicles in the myenteric plexus than did normal diet-fed control mice. Furthermore, mitochondrial volume was increased and lysosome number decreased in the axons of non-treated HFD-fed mice when compared to those of control mice. Phlorizin treatment restored the axonal varicosities and organelles in HFD-fed mice. Although HFD did not affect the immunolocalisation of PGP9.5, it reduced synaptophysin immunostaining in the myenteric plexus, which was restored by phlorizin treatment. These results suggest that impairment of the axonal varicosities and their synaptic vesicles underlies the damage to the enteric neurons caused by HFD feeding. SGLT inhibitor treatment could restore axonal varicosities and organelles, which may lead to improved gastrointestinal functions in HFD-induced obesity as well as diabetes.

Podocyte penetration of the glomerular basement membrane to contact on the mesangial cell at the lesion of mesangial interposition in lupus nephritis: a three-dimensional analysis by serial block-face scanning electron microscopy.

BACKGROUND: The interaction among the glomerular components plays an important role in the development of glomerular lesions; thus, investigation of the ultrastructural three-dimensional (3D) configuration of the human glomerular cells and extracellular matrix (ECM) is important for understanding the pathogenesis of glomerulosclerosis, especially glomerulonephritis. METHODS: We applied a new technique of serial block-face scanning electron microscopy (SBF-SEM), which helps to acquire serial electron microscopic images to reconstruct a 3D ultrastructure, to a human kidney biopsy specimen obtained from a 25-year-old woman with lupus nephritis. RESULTS: SBF-SEM demonstrated that the cytoplasmic processes of the podocyte penetrated into the lamina densa of the glomerular basement membrane, and was in direct contact with the cytoplasm of mesangial cells at the site of mesangial interposition. CONCLUSION: Although this is a single-case observational study, SBF-SEM revealed a unique 3D configuration, suggesting a novel mechanism of direct intercellular cross-communication between podocytes and mesangial cells, aside from the presumed paracrine communication.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets.

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.

Detection of pelagic habitat hotspots for skipjack tuna in the Gulf of Bone-Flores Sea, southwestern Coral Triangle tuna, Indonesia.

Using remote sensing of sea surface temperature (SST), sea surface height anomaly (SSHA) and chlorophyll-a (Chl-a) together with catch data, we investigated the detection and persistence of important pelagic habitat hotspots for skipjack tuna in the Gulf of Bone-Flores Sea, Indonesia. We analyzed the data for the period between the northwest and southeast monsoon 2007-2011. A pelagic hotspot index was constructed from a model of multi-spectrum satellite-based oceanographic data in relation to skipjack fishing performance. Results showed that skipjack catch per unit efforts (CPUEs) increased significantly in areas of highest pelagic hotspot indices. The distribution and dynamics of habitat hotspots were detected by the synoptic measurements of SST, SSHA and Chl-a ranging from 29.5° to 31.5°C, from 2.5 to 12.5 cm and from 0.15 to 0.35 mg m-3, respectively. Total area of hotspots consistently peaked in May. Validation of skipjack CPUE predicted by our model against observed data from 2012 was highly significant. The key pelagic habitat corresponded with the Chl-a front, which could be related to the areas of relatively high prey abundance (enhanced feeding opportunity) for skipjack. We found that the area and persistence of the potential skipjack habitat hotspots for the 5 years were clearly identified by the 0.2 mg m-3 Chl-a isopleth, suggesting that the Chl-a front provides a key oceanographic indicator for global understanding on skipjack tuna habitat hotspots in the western tropical Pacific Ocean, especially within Coral Triangle tuna.

Polymorphic regulation of mitochondrial fission and fusion modifies phenotypes of microglia in neuroinflammation.

Microglia are the resident macrophages of the central nervous system and play complex roles in the milieu of diseases including the primary diseases of myelin. Although mitochondria are critical for cellular functions and survival in the nervous system, alterations in and the roles of mitochondrial dynamics and associated signaling in microglia are still poorly understood. In the present study, by combining immunohistochemistry and 3D ultrastructural analyses, we show that mitochondrial fission/fusion in reactive microglia is differentially regulated from that in monocyte-derived macrophages and the ramified microglia of normal white matter in myelin disease models. Mouse cerebral microglia in vitro demonstrated that stimulation of TLR4 with lipopolysaccharide, widely used to examine microglial reactions, caused the activation of the mitochondrial fission protein, dynamin-related protein 1 (Drp1) and enhanced production of reactive oxygen species (ROS). The increase in the ROS level activated 5' adenosine monophosphate-activated protein kinase (AMPK), and facilitated elongation of mitochondria along the microtubule tracks. These results suggest that the polymorphic regulation of mitochondrial fission and fusion in reactive microglia is mediated by distinct signaling under inflammatory conditions, and modulates microglial phenotypes through the production of ROS.

Inflammatory mediator ultra-low-molecular-weight hyaluronan triggers necrosis of B-precursor leukemia cells with high surface CD44 expression.

Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan (HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging.

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.