Basic designs and recent developments of an atmospheric scanning electron microscope (ASEM) system and optical microscope (OM) are reported. Samples are in liquid or gas at atmospheric pressure on the electron-transparency window of the open ASEM dish which is sealed to the top of an inverted SEM column. OM is placed above the dish. The improved system has (i) a fully motorized sample stage, (ii) a column protection system in case of accidental window breakage, and (iii) an OM/SEM operation system controlled by a graphical user interface. The open sample chamber allows the external administration of reagents to the sample during SEM observation, and the study of systems involving volume changes. Taking advantage of this, we monitored the influence of NaCl on the random motion of silica particles in liquid.
Synapse formation is one of the key steps in the development of neural networks. Precise synaptic connections between nerve cells are crucial for brain development and functions. Recently, several synapse organizers including cerebellar GluRδ2-Cbln1-neurexin triad have been identified. However, their intracellular downstream signals and process of synapse formation are still largely unknown. Atmospheric scanning electron microscope (ASEM) is an electron microscope which was developed recently to directly observe subcellular structures and organelles through a silicon nitride (SiN) film under atmospheric pressure. ASEM dish with the SiN film can be used for incubation of cultured primary neurons. Cultured primary neurons were analyzed by integrated fluoresence microscope and ASEM. Presynaptic differentiation by GluRδ2-NTD-Fc-coated magnetic beads was analyzed by fluoresence microscope and ASEM. Fluorescence signals for presynaptic proteins were accumulated on the beads particularly concentrated at the site of contact with axon. Subsequent ASEM analysis revealed that presynaptic protein, presumably of an indistinguishable neurite branch, surrounded each beads. Thus, ASEM is a potentially useful tool for analysis of molecular localization and fine structure of synapse.
The dynamic features of the cancer cell surface may modulate the malignant phenotype of cancer, including adhesion disorders and aggressive phenotypes of migration. Lipid rafts are cholesterol-enriched microdomains of the cell membrane and possess a highly dynamic nature. They have been involved in various cellular functions including the regulation of cell adhesion and membrane signaling through proteins within lipid rafts. Recently, it was demonstrated that lipid rafts play critical roles in cancer cell adhesion and migration. This article summarizes the important roles of lipid rafts in cancer cell adhesion and migration, with a focus on the current state of knowledge. This article will improve the understanding of cancer progression and lead to the development of novel targets for cancer therapy.
Atmospheric Scanning Electron Microscope (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Primary cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules on microtubules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged.
Recently, swelling and delamination of Layered double hydroxides (LDHs) attract attention for future nano architechnology. The evaluations of swelling and delamination behaviors principally have been carried out using the dry sample by transmission electron microscopy (TEM), powder X-ray diffraction method (XRD) and atomic force microscopy (AFM). But these analyses are not suitable for direct observation of LDHs in solution. In this study, direct observation of swelling and delamination behaviors of LDHs were first carried out in distilled water, sodium laurate solution and formamide solution by Atmospheric Scanning Electron Microscope (ASEM). This new approach method is powerful tool for direct observation of the swelling and delamination behaviors of LDHs in solution.
Nano-structures of aggregates formed by surfactant in aqueous solution can be observed by cryogenic electron microscopy (cryo-TEM). This review describes nano-structures of aggregates by cryo-TEM for three types of novel surfactants with unique structure such as fluorinated gemini-type with two fluorocarbon chains in a molecule, hybrid-gemini-type with nonidentical hydrophobic chains consisting fluorocarbon and hydrocarbon in a molecule, and trimeric-type with three alkyl chains in a molecule. Aggregate structures of these surfactants are differed by alkyl chain length, spacer chain length, and surfactant concentration. These novel surfactants show unique aggregation properties such as polygon vesicle formation for fluorinated gemini-type and formation of multi-lamellar vesicle with large size of 600-800 nm for fluorocarbon-hydrocarbon hybrid-gemini-type.
Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert numerous effects on the central nervous system that regulates the stress response, mood, learning and memory, and various neuroendocrine functions. Corticosterone (CORT) actions in the brain are mediated via two receptor systems: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). These receptors are mainly distributed in the cytoplasm in the absence of hormones and translocated into the nucleus following exposure to hormones to act as transcriptional factors. Thus the subcellular dynamics of both receptors remain important issues under active investigation. Given the differential action of MR and GR in the central nervous system, it is of great interest to clarify how these receptors traffic between cytoplasm and nucleus and how their interactions are regulated by hormones and/or other molecules to exert their transcriptional activity. In this chapter, we describe corticosteroid receptor dynamics in living cells focusing on 1) time-lapse imaging of green fluorescent protein (GFP)-labeled corticosteroid receptors; 2) intranuclear dynamics of GFP-labeled corticosteroid receptors using the fluorescence recovery after photobleaching (FRAP) technique and the fluorescence resonance energy transfer (FRET) technique. We discuss various factors affecting the dynamics of these receptors. Furthermore, we present the future directions of in vivo molecular imaging of corticosteroid receptors at the whole brain level.
A surface plasmon polariton (SPP) is a transverse magnetic (TM) mode electromagnetic wave propagating at a metal/dielectric interface, which is evanescently confined in the perpendicular direction. Metallic surfaces with periodic structures on a sub-wavelength scale have recently been called “plasmonic crystals (PlCs)” in the developing field of plasmonics. We investigate the properties of SPP in plasmonic crystals by using cathodoluminescence (CL) technique combined with a 200 keV scanning transmission electron microscope (STEM). The SPPs excited by high energy electrons are converted to light when propagating on PlCs. Angle resolved spectral measurement of the emitted light enables us to deduce a dispersion pattern of SPP on PlCs, and a beam scan spectral image reveals a standing SPP wave of the band edge state. Size dependence of band gap and symmetry of the standing SPPs were investigated for 1D and 2D PlCs with changing shape parameters.
Meso-structured materials have structural characteristics in meso-scale, and mesoporous silica is one of the meso-structured materials. For structural characterization of mesoporous silicas, transmission electron microscopy (TEM) is crucial, since they have two defining structural characteristics, i.e. disorder on the atomic-scale and distinct order on the meso-scale. The following topics are discussed in this article. (i) three-dimensional (3D) structural characterization of mesoporous silicas by electron crystallography. (ii) 3D mesopore structures inside spherical silica nanoparticles and bicontinuous mesostructures characterized by electron tomography, and the combination of electron tomography and HAADF-STEM. (iii) structural characterization of binary nano-colloidal crystals by electron microscopy.
For endoparasitic organisms, entering the host body is essential. During the immature stage of its life cycle, the insect parasitoid Copidosoma floridanum feeds onanother insect. The morula-stage embryos of C. floridanum can move like amoebae and actively invade host embryos by using molecular mimicry. Our previous study showed that cadherins and C-type lectins are involved in this phenomenon. This is the first report showing a receptor–ligand interaction between heterologous multicellular organisms. Electron microscopy analyses of cellular interactions between the extraembryonic syncytium of invading parasitic morula and the host embryonic epithelial cells clearly showed that morula penetration into the host embryo causes no damage to the host cells. Epithelial cells of the host embryo extended microvilli toward invading C. floridanum morula and adjacent host cells in the same way. Shortly after settlement of the morula within the host body cavity, small gap junctions and adherens junctions with host cells were formed. The morula was then surrounded by a cyst comprised of host cells into which host tracheoles were invaginated.
The concept and recent progress of three-dimensional reconstruction observation by means of a focused ion beam (FIB)-scanning electron microscope (SEM) serial sectioning method is described. The characteristic points of the orthogonally-arranged FIB-SEM is introduced. This new equipment is specially designed to improve the quality of serial sectioning observation: it shows high contrast and high spatial resolution. These features are realized by the ideal configuration of the SEM, FIB and other detectors such as the short working distance (2 mm). From the results carried out by this FIB-SEM, methodology and quality of observation are discussed.
Fluorescent microscopy techniques allow to obtain multicolor cellular images by using a variety of fluorescent probes which can selectively visualize molecular species. It is expected that electron microscopy could generate color images of distinct single molecules with high spatial resolution. We synthesized rare-earth doped Y2O3 nanophosphors which emit cathodoluminescence (CL) in 3 different colors under electron beam excitation. These nanophosphores emit fluorescence by UV light excitation which indicates their potential for fluorescent imaging as well. In this manuscript, we explain the synthesis method and applications of nanophosphors using electron and fluorescent microscopy.
We review a non-commercial environmental scanning electron microscope AQUASEM II that have been developed in Institute of Scientific Instruments of the ASCR, Czech Republic. AQUASEM II is equipped with four electron detectors, which are a YAG:Ce3+ scintillation single crystal detector with high efficiency for backscattered electrons and (i) a newly patented ionization secondary electron detector with electrostatic separator (ISEDS), (ii) an ionization secondary electron detector and (iii) a newly designed scintillation secondary electron detector for SEM and ESEM. AQUASEM II, which is equipped with a specially designed differentially pumping and hydration system for the specimen chamber as well as the electron signal detection system of four detectors, have allowed us to perform in-situ observation or in-situ dynamical observation of samples such as semiconductors in problematic conditions and live biological samples.
Scanning electron microscopy (SEM) has made remarkable progress, and has become an essential tool for observing the biological materials. However, various complex procedures preclude observation of living organisms to date. We reported previously that the simple surface modification gives rise to a thin extra layer, which we coined “NanoSuit”, and hence can keep them alive in the high vacuum (10-5-10-7 Pa) of a field emission (FE) SEM. In this research, to examine further the function of the NanoSuit, we investigated various raw specimens simply protected by NanoSuit in the FE-SEM. In addition to seeing spontaneous movements, we found that the surface fine structure of the living organism is very different from that of traditionally fixed samples. From these observations, we anticipate our findings to be the starting point for more sophisticated observation of living organisms with the electron microscope and for the creation of new areas of biology, chemistry and physics in order to explain how the NanoSuit forms a gas and/or liquid barrier based on the Surface Shield Effect (SS-effect) to preserve life under a vacuum.