Human induced pluripotent stem cells are resistant to human cytomegalovirus infection primarily at the attachment level due to the reduced expression of cell-surface heparan sulfate.

Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level. IMPORTANCE: Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo. In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted.

Route of pesticide spread on the body surface of Blattella germanica (Linnaeus): a NanoSuit-energy dispersive X-ray spectroscopy analysis.

Numerous studies have focussed on the mechanisms of entry of pesticides into insect body parts such as oral intake, penetration through the integument of the body wall, and inhalation through spiracles. However, little is known about how insecticides spread to the points of entry or the paths on the body surface that are used to reach the target sites. In this study, elemental signals of pesticide-mimicking test solutions were tracked and their routes of spreading in experimental insects (Blattella germanica L.) were investigated using NanoSuit (a method of surface modification) and energy dispersive X-ray spectroscopy, combined with high-resolution scanning electron microscopy. When the test solution initially adhered to the dorsal and/or ventral body surface, it tended to spread horizontally to reach lateral plates. Whereas, when the solution directly adhered to the anterior side of the lateral plates, it spread to posterior segments. In this case, however, spreading in the opposite direction (i.e., the solution directly adhered to the posterior side of the lateral plates) was interrupted at a boundary erected by different groups of fine structures; each protrusion was large, and the arrangement was rather dense in the posterior segments. Morphological features of these fine structures and chemical characteristics of the hydrophobic surface substances potentially regulate the strength of the capillary force, which determines pesticide spreading.

The use of polarized light in the zonal orientation of the sandhopper Talitrus saltator (Montagu).

It is well known that the celestial polarization is used as a compass cue by many species of insects and crustaceans. Although it has been shown that the sandhopper Talitrus saltator perceives polarized light and possesses an arrangement of the rhabdomeres that could allow e-vector interpretation and utilization, T. saltator does not use the e-vector of the skylight polarization as a compass cue when making excursions along the sea-land axis of sandy shores. We performed tests in confined conditions to clarify if skylight polarization is somehow involved in the zonal recovery of T. saltator. We observed the directional responses of sandhoppers in a transparent bowl under an artificial sky (an opaline Plexiglas dome). The bowl was covered by a blue gelatin filter with a grey filter (control condition) and a linear polarizing filter (experimental conditions) positioned under the blue one in such a way as to occupy half of the upper surface of the Plexiglas bowl so as to create a linear polarization gradient. Our experiments confirm that T. saltator perceives polarized light and highlight that this visual capability determines the perception, or perhaps the increase, of the radiance and/or spectral gradient and their use as compass cues in the zonal orientation. Moreover, our findings confirm that the radiance gradient is used as a chronometric compass orienting reference in the absence of other celestial orienting cues.

Primary Cilia Are Frequently Present in Small Cell Lung Carcinomas but Not in Non-Small Cell Lung Carcinomas or Lung Carcinoids.

Most human malignant neoplasms show loss of primary cilia (PC). However, PC are known to be retained and involved in tumorigenesis in some types of neoplasms. The PC status in lung carcinomas remains largely uninvestigated. In this study, we comprehensively assessed the PC status in lung carcinomas. A total of 492 lung carcinomas, consisting of adenocarcinomas (ACs) (n = 319), squamous cell carcinomas (SCCs) (n = 152), and small cell lung carcinomas (SCLCs) (n = 21), were examined by immunohistochemical analysis using an antibody against ARL13B, a marker of PC. The PC-positive rate was markedly higher in SCLCs (81.0%) than in ACs (1.6%) and SCCs (7.9%). We subsequently performed analyses to characterize the PC-positive lung carcinomas further. PC-positive lung carcinomas were more numerous and had longer PC than normal cells. The presence of PC in these cells was not associated with the phase of the cell cycle. We also found that the PC were retained even in metastases from PC-positive lung carcinomas. Furthermore, the hedgehog signaling pathway was activated in PC-positive lung carcinomas. Because ARL13B immunohistochemistry of lung carcinoids (n = 10) also showed a statistically significantly lower rate (10.0%) of PC positivity than SCLCs, we searched for a gene(s) that might be upregulated in PC-positive SCLCs compared with lung carcinoids, but not in PC-negative carcinomas. This search, and further cell culture experiments, identified HYLS1 as a gene possessing the ability to regulate ciliogenesis in PC-positive lung carcinomas. In conclusion, our findings indicate that PC are frequently present in SCLCs but not in non-SCLCs (ACs and SCCs) or lung carcinoids, and their PC exhibit various specific pathobiological characteristics. This suggests an important link between lung carcinogenesis and PC.

Association between human papillomavirus particle production and the severity of recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) has a wide range of severity. We investigate the relationship between human papillomavirus (HPV) particle production and severity of RRP. From September 2005 to June 2021, 68 RRP samples (from 29 patients) were included. HPV type was determined. HPV viral load, physical status, and demographic and clinical characteristics were assessed. Immunohistochemistry (IHC) was performed for p16, Ki-67, L1, and E4. We used NanoSuit-CLEM (correlative light and electron microscopy) and transmission electron microscopy (TEM) to examine the samples. The total number of surgeries in HPV-positive and HPV-negative cases were 3.78 (n = 55/68, range: 1-16) and 1.30 (n = 13/68, range: 1-3), respectively (p = 0.02). IHC showed that L1 and E4 were correlated and expressed on the tumour surface. NanoSuit-CLEM and TEM revealed HPV particles in L1-positive nuclei. L1 IHC-positive cases had a shorter surgical interval (p < 0.01) and more frequent surgeries (p = 0.04). P16 IHC, viral load, and physical status were not associated with disease severity. This study visualised HPV particle production in RRP for the first time. Persistent HPV particle infection was associated with severity. We suggest L1 IHC for evaluating RRP severity in addition to the Derkay score.

Identifying Active Progeny Virus Particles in Formalin-Fixed, Paraffin-Embedded Sections Using Correlative Light and Scanning Electron Microscopy.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.

Highly Sensitive and Quantitative Diagnosis of SARS-CoV-2 Using a Gold/Platinum Particle-Based Lateral Flow Assay and a Desktop Scanning Electron Microscope.

The gold standard test for identifying SARS-CoV-2, the causative agent of COVID-19, is polymerase chain reaction (PCR). Despite their limited sensitivity, SARS-CoV-2 antigen rapid diagnostic tests are vital tools in the fight against viral spread. Owing to its simplicity and low cost, the lateral flow assay (LFA) is the most extensively used point-of-care diagnostic test. Here, we report a newly designed LFA-NanoSuit method (LNSM) that works in conjunction with desktop scanning electron microscopy (SEM) to detect SARS-CoV-2. LNSM requires no standard SEM treatment, avoids cellulose and residual buffer deformation, and enables the capture of high-resolution images of antibody-labeled gold/platinum particles reacting with SARS-CoV-2 antigens. To assess its applicability, we compared clinical SARS-CoV-2 samples via visual detection of LFA, LSNM detection of LFA, and real-time reverse transcription-PCR (qRT-PCR). Compared to qRT-PCR, LNSM showed 86.7% sensitivity (26/30; 95% confidence interval (CI): 69.28-96.24%) and 93.3% specificity (14/15; 95% CI: 68.05-99.83%) for SARS-CoV-2. In samples with a relatively low SARS-CoV-2 RNA copy number (30 < Ct ≤ 40), the sensitivity of LNSM was greater (73.3%) than that of visual detection (0%). A simple, sensitive, and quantitative LNSM can be used to diagnose SARS-CoV-2.

Microscopy and biomimetics: the NanoSuit® method and image retrieval platform.

This review aims to clarify a suitable method towards achieving next-generation sustainability. As represented by the term 'Anthropocene', the Earth, including humans, is entering a critical era; therefore, science has a great responsibility to solve it. Biomimetics, the emulation of the models, systems and elements of nature, especially biological science, is a powerful tool to approach sustainability problems. Microscopy has made great progress with the technology of observing biological and artificial materials and its techniques have been continuously improved, most recently through the NanoSuit® method. As one of the most important tools across many facets of research and development, microscopy has produced a large amount of accumulated digital data. However, it is difficult to extract useful data for making things as biomimetic ideas despite a large amount of biological data. Here, we would like to find a way to organically connect the indispensable microscopic data with the new biomimetics to solve complex human problems.

Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

BACKGROUND AND OBJECTIVE: Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. METHODS: Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (µCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. RESULTS: The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. CONCLUSIONS: HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

Antenna Cleaning Is Essential for Precise Behavioral Response to Alarm Pheromone and Nestmate-Non-Nestmate Discrimination in Japanese Carpenter Ants (Camponotus japonicus).

Self-grooming of the antennae is frequently observed in ants. This antennal maintenance behavior is presumed to be essential for effective chemical communication but, to our knowledge, this has not yet been well studied. When we removed the antenna-cleaning apparatuses of the Japanese carpenter ant (C. japonicus) to limit the self-grooming of the antennae, the worker ants demonstrated the self-grooming gesture as usual, but the antennal surface could not be sufficiently cleaned. By using scanning electron microscopy with NanoSuit, we observed the ants' antennae for up to 48 h and found that the antennal surfaces gradually became covered with self-secreted surface material. Concurrently, the self-grooming-limited workers gradually lost their behavioral responsiveness to undecane-the alarm pheromone. Indeed, their locomotive response to the alarm pheromone diminished for up to 24 h after the antenna cleaner removal operation. In addition, the self-grooming-limited workers exhibited less frequent aggressive behavior toward non-nestmate workers, and 36 h after the operation, approximately half of the encountered non-nestmate workers were accepted as nestmates. These results suggest that the antennal sensing system is affected by excess surface material; hence, their proper function is prevented until they are cleaned.

Diagnosis of Ion-Exchange Resin Depositions in Paraffin Sections Using Corrective Light and Electron Microscopy-NanoSuit Method.

Ion-exchange resins are commonly used to treat complications such as hyperkalemia, hyperphosphatemia, and hypercholesterolemia. Gastrointestinal complications may occur as side effects of such treatments. Sodium and calcium polystyrene sulfonate (PS-Ca) are cation-exchange resins comprising an insoluble structure that binds to potassium ions in the digestive tract and exchanges them with sodium and calcium ions, respectively, to promote their elimination. PS crystals are rhomboid, refractive, and basophilic in hematoxylin and eosin staining. To differentiate PS crystals from other ion-exchange resin crystals such as sevelamer and cholestyramine, periodic acid-Schiff, Ziehl-Neelsen, and Congo red staining are usually performed. Here, correlative light and electron microscopy (CLEM)-energy-dispersive X-ray spectroscopy and the NanoSuit method (CENM) was applied to perform a definitive identification of ion-exchange resins. CENM could detect sulfur in PS crystals without destroying the glass slides. Notably, PS retained its ion-exchange ability to bind potassium in paraffin sections. Differential diagnosis of anion-exchange resins, such as sevelamer and cholestyramine, was possible using these characteristics. The phosphorus:carbon ratio was higher in sevelamer than in cholestyramine after soaking paraffin sections in a phosphate solution. Therefore, CENM may be used for the differential pathological diagnosis of ion-exchange resins in paraffin sections.

Hydrophobic-hydrophilic crown-like structure enables aquatic insects to reside effectively beneath the water surface.

Various insects utilise hydrophobic biological surfaces to live on the surface of water, while other organisms possess hydrophilic properties that enable them to live within a water column. Dixidae larvae reside, without being submerged, just below the water surface. However, little is known about how these larvae live in such an ecological niche. Herein, we use larvae of Dixa longistyla (Diptera: Dixidae) as experimental specimens and reveal their characteristics. A complex crown-like structure on the abdomen consists of hydrophobic and hydrophilic elements. The combination of these contrasting features enables the larvae to maintain their position as well as to move unidirectionally. Their hydrophobic region leverages water surface tension to function as an adhesive disc. By using the resistance of water, the hydrophilic region serves as a rudder during locomotion.

Identification and characterization of primary cilia-positive salivary gland tumours exhibiting basaloid/myoepithelial differentiation.

Primary cilia (PC) are non-motile, antenna-like structures on the cell surface. Many types of neoplasms exhibit PC loss, whereas in some neoplasms PC are retained and involved in tumourigenesis. To elucidate the PC status and characteristics of major salivary gland tumours (SGTs), we examined 100 major SGTs encompassing eight histopathological types by immunohistochemical analysis. PC were present in all (100%) of the pleomorphic adenomas (PAs), basal cell adenomas (BCAs), adenoid cystic carcinomas (AdCCs), and basal cell adenocarcinomas (BCAcs) examined, but absent in all (0%) of the Warthin tumours, salivary duct carcinomas, mucoepidermoid carcinomas, and acinic cell carcinomas examined. PC were also detected by electron-microscopic analysis using the NanoSuit method. It is worthy of note that the former category and latter category of tumours contained and did not contain a basaloid/myoepithelial differentiation component, respectively. The four types of PC-positive SGTs showed longer PC than normal and exhibited a characteristic distribution pattern of the PC in the ductal and basaloid/neoplastic myoepithelial components. Two PC-positive carcinomas (AdCC and BCAc) still possessed PC in their recurrent/metastatic sites. Interestingly, activation of the Hedgehog signalling pathway, shown by predominantly nuclear GLI1 expression, was significantly more frequently observed in PC-positive SGTs. Finally, we identified tau tubulin kinase 2 (TTBK2) as being possibly involved in the production of PC in SGTs. Taken together, our findings indicate that SGTs that exhibit basaloid/myoepithelial differentiation (PA, BCA, AdCC, and BCAc) are ciliated, and their PC exhibit tumour-specific characteristics, are involved in activation of the Hedgehog pathway, and are associated with TTBK2 upregulation, providing a significant and important link between SGT tumourigenesis and PC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Combination of the NanoSuit method and gold/platinum particle-based lateral flow assay for quantitative and highly sensitive diagnosis using a desktop scanning electron microscope.

Owing to its simplicity and low cost, the lateral flow assay (LFA) is one of the most commonly used point-of-care diagnostic techniques, despite its low sensitivity and poor quantification. Here, we report a newly developed LFA-NanoSuit method (LNSM) combined with a desktop scanning electron microscope (SEM) for the direct observation of immunocomplexes labeled with a colloidal metal instead of signal enhancement strategies, such as using color, electrochemical signals, silver enhancement, magnetic properties, luminescent, and surface-enhanced Raman spectroscopy (SERS). The proposed LNSM suppresses cellulose deformity, thereby allowing the acquisition of high-resolution images of gold/platinum-labeled immunocomplexed pathogens such as influenza A, without conductive treatment as in conventional SEM. Electron microscopy-based diagnosis of influenza A exhibited 94 % clinical sensitivity (29/31; 95 % confidence interval [CI]: 79.3-98.2 %) and 100 % clinical specificity (95 % CI: 98.1-100 %), which was more sensitive (71.4 %) than visual detection (14.3 %), especially in the lower influenza A-RNA copy number group. The detection ability of our method was nearly comparable to that of real-time reverse transcription-PCR. This is the first report on the diagnosis of clinical diseases using LFA equipped with a desktop SEM. This simple and highly sensitive quantitative analysis method involving LFA can be used to diagnose various diseases in humans and livestock, including highly infectious diseases such as COVID-19.

Synergistic Effects of the Red Light and Blue Traps on Control of Thrips palmi (Thysanoptera: Thripidae).

The melon thrips (Thrips palmi) is a serious insect pest of vegetables and ornamental plants. To control and monitor this thrips, blue traps are widely used. We previously reported that irradiation of cucumber plants with red light-emitting diodes (660 nm) causes T. palmi to avoid the plants. Here, we evaluated the responses of T. palmi adults to a cucumber plant in an experimental arena with either a transparent sticky trap or a blue sticky trap, without or with illumination by a red LED panel. When T. palmi were introduced into the arena, the number of thrips attracted to the plant was lowest in treatments using the red light and blue trap. On the other hand, when T. palmi were inoculated on plants first, most thrips stayed put on the plants. Placing both red LEDs and blue sticky traps in greenhouses before the thrips invade the greenhouse from the outside might effectively control T. palmi.

Sky radiance and spectral gradient are orienting cues for the sandhopper Talitrus saltator (Crustacea, Amphipoda).

The sandhopper Talitrus saltator relies on both the sun and the moon compasses to return to the belt of damp sand on the beach in which it lives buried during the day. In addition to the sun, the gradient of radiance and the spectral distribution across the sky could provide directional information that T. saltator can potentially use to orient itself during the day even when the sun is not visible (e.g. cloudy sky). The scope of this work was (1) to determine the intensity levels of sky radiance that the sandhoppers use in their zonal recovery and (2) to investigate whether this species relies on the celestial spectral gradient in its zonal recovery. Sandhoppers were tested in the laboratory under artificial radiance or spectral gradients. Our results show that under an artificial sky simulating the natural radiance gradient on a cloudless day, sandhoppers orientated toward the correct seaward direction of their home beach; however, individuals lost their ability to use the intensity gradient as an orientation cue when the radiance was attenuated by at least 40%. Sandhoppers were also able to head in the correct seaward direction of their home beach at any time of the day by using the spectral gradient as their only source of visual orientation reference.

Visual adaptation of opsin genes to the aquatic environment in sea snakes.

BACKGROUND: Evolutionary transitions from terrestrial to aquatic life history cause drastic changes in sensory systems. Indeed, the drastic changes in vision have been reported in many aquatic amniotes, convergently. Recently, the opsin genes of the full-aquatic sea snakes have been reported. However, those of the amphibious sea snakes have not been examined in detail. RESULTS: Here, we investigated opsin genes and visual pigments of sea snakes. We determined the sequences of SWS1, LWS, and RH1 genes from one terrestrial, three amphibious and four fully-aquatic elapids. Amino acid replacements at four and one spectra-tuning positions were found in LWS and RH1, respectively. We measured or predicted absorption of LWS and RH1 pigments with A1-derived retinal. During their evolution, blue shifts of LWS pigments have occurred stepwise in amphibious sea snakes and convergently in both amphibious and fully-aquatic species. CONCLUSIONS: Blue shifted LWS pigments may have adapted to deep water or open water environments dominated by blue light. The evolution of opsins differs between marine mammals (cetaceans and pinnipeds) and sea snakes in two fundamental ways: (1) pseudogenization of opsins in marine mammals; and (2) large blue shifts of LWS pigments in sea snakes. It may be possible to explain these two differences at the level of photoreceptor cell composition given that cone and rod cells both exist in mammals whereas only cone cells exist in fully-aquatic sea snakes. We hypothesize that the differences in photoreceptor cell compositions may have differentially affected the evolution of opsins in divergent amniote lineages.

In situ elemental analyses of living biological specimens using 'NanoSuit' and EDS methods in FE-SEM.

Energy dispersive X-ray spectroscopy (EDS) carried out alongside scanning electron microscopy (SEM) is a common technique for elemental analysis. To investigate "wet" biological specimens, complex pre-treatments are required to stabilize them under the high vacuum conditions of high-resolution SEM. These often produce unwanted artifacts. We have previously reported that the polymerization of natural surface substances on organisms by the electron beam of the SEM setup or by plasma irradiation causes a nano-scale layer to form-called a "NanoSuit"-that can act as a barrier and keep organisms alive and hydrated in a field-emission SEM system. In the study reported herein, we examined the suitability of the NanoSuit method for elemental analyses of biological specimens by EDS. We compared experimental results for living Drosophila larvae and Aloe arborescens specimens prepared by the NanoSuit method and by conventional fixation. The NanoSuit method allowed accurate detection of the elemental compositions at high resolution. By contrast, specimens prepared by the conventional fixation method displayed additional EDS signals corresponding to the elements in the chemicals involved in the fixation process. Our results demonstrate that the NanoSuit method is useful for studying hydrous samples via EDS and SEM, particularly in biological sciences.

Specializations in the compound eye of Talitrus saltator (Crustacea, Amphipoda).

We investigated the eye regionalization in Talitrus saltator by morphological, electrophysiological and behavioural experiments. Each ommatidium possesses five radially arranged retinular cells producing a square fused rhabdom by R1-R4 cells; the smaller R5 exists between R1 and R4. The size of R5 rhabdomere is larger in the dorsal part and becomes smaller in the median and ventral parts of the eye. Spectral-sensitivity by electroretinograms were recorded from dorsal or ventral parts of the eye. The dorsal part possesses maxima at green and UV-blue region. The main response region in the ventral part is only from UV (390 nm) to blue (430 nm) decreasing at longer wavelengths. To evaluate the sandhoppers' celestial orientation, their eyes were painted black either in the dorsal or ventral part, under the natural sky or a blue filter with or without the vision of the sun. Sandhoppers with the dorsal region of the eyes painted and tested under the screened sun were more dispersed and their directions varied more than in other groups of individuals. Sandhoppers with this area of the eye obscured display considerable difficulties to head in a specific direction. This work suggests the existence of regional specializations in the eye of T. saltator.

Imaging dataset of fresh hydrous plants obtained by field-emission scanning electron microscopy conducted using a protective NanoSuit.

Although scanning electron microscopy (SEM) can generate high-resolution images of nanosized objects, it requires a high vacuum to do so, which precludes direct observations of living organisms and often produces unwanted structural changes. It has previously been reported that a simple surface modification gives rise to a nanoscale layer, termed the "NanoSuit", which can keep small animals alive under the high vacuum required for field-emission scanning electron microscopy (FE-SEM). We have previously applied this technique to plants, and successfully observed healthy petals in a fully hydrated state using SEM. The flower petals protected with the NanoSuit appeared intact, although we still lack a fundamental understanding of the images of other plants observed using FE-SEM. This report presents and evaluates a rich set of images, acquired using the NanoSuit, for a taxonomically diverse set of plant species. This dataset of images allows the surface features of various plants to be analyzed and thus provides a further complementary morphological profile. Image data can be accessed and viewed through Figshare (https://doi.org/10.6084/m9.figshare.c.4446026.v1).