Although electron tomography has been rapidly developed, it is still difficult to analyze fine structures such as protein complexes in the cell. It is mainly because of two reasons: 1. Poor signal to noise ratio in the cell because of cytosol. 2. Missing wedge or missing pyramid caused by limited tilt angles of the sample deform the reconstructed three-dimensional map. We have developed structural analysis methods for filamentous complexes, especially for actin filaments in the cell. John Victor Small’s group, our co-researchers, has enhanced the signal to noise ratio by staining the cell negatively. We have developed an image analysis system for analyzing electron tomograms of the negatively stained cells, which enabled us to determine polarity of the actin filaments without any labeling. We review these methods in this article.
Fluorescence single-molecule speckle microscopy is a powerful tool to elucidate intracellular dynamics of cytoskeleton-associated molecules. However, it is often difficult to precisely understand how the observed molecule behave as a whole in the system. On the other hand, imaging approaches such as FRAP (fluorescence recovery after photobleaching), which visualize redistribution of bulk population of the molecule, has limited spatiotemporal resolution, leading to imperfect interpretation by overlooking a minor population of molecular species with distinct kinetics. We will introduce our recent studies in which combination analysis of fluorescence single-molecule imaging with FRAP or s-FDAP (sequential-fluorescence decay after photoactivation) provided better understanding in the regulation of actin polymerization and depolymerization cycles.
The mitotic spindle is a highly organized microtubule-based structure that is required for accurate chromosome segregation. It is generally known that the centrosomes and the chromosomes function as scaffolds for microtubule generation during spindle formation. In addition, emerging evidence suggests that the microtubules within the spindle are important for the spindle microtubule supplementation. An eight-subunit protein complex augmin plays a pivotal role in the microtubule-dependent microtubule generation during spindle formation, but the molecular basis of augmin’s functions remains unclear. Our latest research, which uses electron tomography and 3-D modeling, revealed that microtubule branching occurs within the spindle in an augmin-dependent fashion, demonstrating the ultrastructural basis of the microtubule-dependent microtubule generation during functional spindle formation.
Cilia and flagella are elaborate organelles found in a wide range of eukaryotes. The beating motion of flagella is generated by hundreds of axonemal proteins, but functional analyses of individual molecules were insufficient to reveal the complex regulatory mechanism. Chlamydomonas, one species of green algae, is a precious model organism in that various experimental methods such as biochemical, genetical, cell biological and electron microscopic analyses have been established by decades of research. We took advantage of this characteristic of Chlamydomonas and have found the regulatory mechanism for the axonemal motor protein, dynein.
Ionic liquids are salts in liquid state even at room temperature. Since they have extremely low vapor pressure, they are not vaporized even in vacuum conditions. Focusing on this fact, the author has discovered that ionic liquids can be observed by scanning electron microscope without any charging, implying that ionic liquids behave as conducting materials for electron scope observations. Then, the author is now developing new electron scope techniques by using ionic liquids as liquid conductive materials. The use of ionic liquids is very effective for the SEM observation of soft bio-samples whose surfaces are very complex and are changed by drying. In case of the TEM observation, forming liquid films in the holes of a TEM grid and putting samples in the liquid films enable the observation of samples without any deformation. When chemical reactions are induced in an ionic liquid that is placed in a SEM chamber, in situ SEM observation and in situ EDX measurement of the reactions are possible. Some examples of electrochemical reactions such as metal deposition, metal dissolution, and electrochemical actuator reactions are introduced in this article.
High-resolution observation of cells and tissues is principally carried out by electron microscopy (EM), although standard EM requires the sample be in vacuum. In the ASEM, an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized a dynamic string-like gathering of STIM1 on the ER in Jurkat T cells in response to Ca2+ store depletion. We have also visualized filamentous-actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca2+ signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework.
The goal of specimen preparation for transmission electron microscopy is to obtain high–quality ultra-thin sections with which we can correlate cellular structure to physiological function. In practice, prior to ultra-thin sectioning, semi-thin sections are usually examined to screen the sample quality at light microscopic level. The screened semi-thin sections are utilizable to obtain the desired ultra-thin sections after re-embedding into the same resin. Recently, we have developed a new capsule-supporting ring useful for re-embedding of semi-thin sections to be cut into ultrathin sections for electron microscopy. The capsule-supporting ring, punched out from a sheet of ethylene-vinyl acetate (EVA) resin foam, is attached with adhesive tape on one face which prevents leakage of resin and also occasional sliding of the capsule during polymerization of resin. For electron microscopy of the cultured cells, the present method enables us to culture, fix, and embed the cells in situ cultivated on a coverslip, preserving ultrastructures close to their native state. The capsule-supporting ring is also applicable to low viscous hydrophilic resins, such as Lowicryl series, useful for gold labeling. We practically describe this new preparation technique for targeting ultrastuctural observation by means of the capsule-supporting ring.
As an active nano characterization technology, we developed an atomic resolution ultrahigh vacuum dual probe scanning probe microscope with an in-situ external stress application capability in order to determine the effects of stress and strain fields on surface atomistic structures. It is necessary to understand these effects because controlling them will be a key technology that will very likely be used in future nanometer-scale fabrication processes. We used our stress-strain filed scanning probe microscope (SF-SPM) to demonstrate atomic resolution imaging under external tensile stress and strain on the surfaces of wafers of Si(111) and Si(001). The developed SF-SPM has two operation mode, scanning tunneling microscopy (STM) and noncontact atomic force microscopy (NCAFM) modes with atomic resolution imaging. We successfully observed domain redistribution induced by applying uniaxial stress at an elevated temperature on the surface of a wafer of vicinal Si(100). We discovered that domains for which an applied tensile stress is directed along the dimer bond become less stable and shrink. This suggests it may be feasible to fabricate single domain surfaces in a process that controls surface stress and strain.
There are only two kinds of organisms on earth: prokaryotes and eukaryotes. Although eukaryotes are considered to have evolved from prokaryotes, there were no previously known intermediate forms between them. The differences in their cellular structures are so vast that the problem of how eukaryotes could have evolved from prokaryotes is one of the greatest enigmas in biology. Here we report a unique organism with cellular structures appearing to have intermediate features between prokaryotes and eukaryotes that was discovered in the deep-sea off the coast of Japan by using electron microscopy. The organism was 10 μm long and 3μm in diameter, having more than 100 times volume of Escherichia coli. It had a large ‘nucleoid’, consisting of naked DNA fibers, with a single layered ‘nucleoid’ membrane, and endosymbionts that resemble bacteria, but no mitochondria. Because this organism as appears to be a life form distinct from both prokaryotes and eukaryotes but similar to eukaryotes, we named this unique microorganism the ‘Myojin parakaryote’ with the scientific name of Parakaryon myojinensis (“next to (eu)karyote from Myojin”) after the discovery location and its intermediate morphology. The existence of this organism is an indication of a potential evolutionary path between prokaryotes and eukaryotes.
To measure a fine electronic structure in EELS for a specimen at high spatial and energy resolutions, we have developed a new analytical electron microscope equipped with a monochromator, which incorporates a double Wien-filter system. The electron probe was resulted to be highly monochromated and roundly shaped on the specimen plane. The ultimate energy resolutions, measured in FWHMs, for zero-loss peaks were measured to be 36 meV at 200 kV and 30 meV at 60 kV with 0.1 seconds acquisitions. In the experiment of EELS mapping, the map showed an atomic resolution and the spectrum showed an energy resolution of 146 meV.