The combination of focused ion-beam milling with scanning electron microscopy (SEM) backscattered imaging provides a versatile three-dimensional (3D) reconstruction method (FIB/SEM tomography) that can visualize the hierarchical structural organization of tissue, cell and organelle with a similar resolution in electron microscopy. The nanofabrication technology of FIB allows milling of various solid materials—including bone tissue, which is one of difficult material to cut off with mechanical methods—and following 3D reconstruction of the specimen at resolution as good as that of an electron tomography (>5 nm). Although the possible size of the 3D reconstruction is limited to tens of micrometers cubic, the survey specimen sizes are not limited. Thus, 3D reconstruction targets can be selected by an overview of a large specimen area. This procedure is useful for biomedical materials as well as clinical subjects and the correlative observation of light and electron microscopy. The method is thus indispensable for in imaging of cell and tissue architectures in their entirety at nanometer-range resolution.
Serial block face-scanning electron microscopy (SBF-SEM) repeats milling of tissue blocks by built-in ultramicrotome and subsequent observation of the tissue block surface with SEM. It facilitates rapid acquisition of serial electron microscopic images at a resolution of several nm from tissue areas larger than hundreds μm2. This SEM approach requires adequate en bloc electron staining and also optimization of embedding, trimming and final observation conditions to reduce a specimen charging, increase contrast and stably acquire images of cells and tissues. Because of its rapid image acquisition, SBF-SEM is useful for high-throughput morphological analyses of various disease models and transgenic animals as well as “connectome” analyses in large tissue areas of the nervous system. Furthermore, its combination with molecular labeling can detect specific organelles and cells in 3-dimensional tissue architectures. Therefore, SBF-SEM will be widely used as a new approach for 3-dimensional analyses of ultrastructures in biological specimens which have been difficult to observe with conventional transmission electron microscopy.
Serial section scanning electron microscopy (SEM) is based on the collection of backscattered electron images of serial ultrathin sections stained with heavy metals. This method is simpler than serial section transmission electron microscopy, and is useful for 3D reconstruction of a region of interest without using special instruments such as FIB/SEM (focused ion beam/SEM) or SBF/SEM (serial block face/SEM). In this paper, we will explain details of serial section SEM, and show its application for 3D morphological analysis of the Golgi apparatus. This technique is suitable for 3D morphological reconstruction of the Golgi apparatus which is known as a cell organelle with a complicated structure. Serial section SEM is expected to be widely used for analysis of 3D structure of such cell organelle as Golgi apparatus and mitochondria.
Recently, technologies for three-dimensional reconstruction of biological samples have developed rapidly. Especially, various methods using scanning electron microscopy have emerged. ATUM (Automated Tape-Collecting system Ultra-Microtome) is one of these technologies and ultrathin sections are collected automatically by using ATUM. In this article, I will introduce the detail of ATUM and will describe how to use this equipment, including the application for light microscopy.
Crystal structures of long-period stacking-ordered (LPSO) phases in the Mg-TM (transition-metal) -RE(rare-earth) systems were investigated by atomic resolution high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and transmission electron microscopy (TEM). The 18R-type LPSO phase in the Mg-Al-Gd system is composed by stacking structural blocks with a fully-ordered atomic arrangement. However, the stacking of structural blocks does not exhibit any long-range order along the stacking direction. Because of these characteristics, the LPSO phase in the Mg-Al-Gd system cannot be described as an ‘LPSO’ phase any longer in a strict sense but as an order-disorder (OD) intermetallic phase with a so-called OD structure. Details of the crystal structure analysis of the Mg-Al-Gd LPSO phases are described on the basis of the OD theory.
Tract tracing is one of the central topics of neuroanatomy. To elucidate the cells of origin, routes, and terminals of neural pathways, a variety of techniques have been developed including analysis of myelogenesis and demyelination, staining for degenerating myelin and axons, Golgi methods, and tracing using axonal transport and viral infections. To visualize normal morphology of a neural pathway, it is necessary to make constituent neurons incorporate as much reporter molecules as possible. Recent gene transfer techniques fulfill this requirement by making neurons produce reporter molecules themselves. Tract tracing using gene transfer is now combined with the control of neuronal activities by transgenes, and opened a new field of comprehensive analysis of morphology and functions of neural pathways.
The three-dimensional structure of mitochondria within the rat’s olfactory-bulb granule cells was investigated using high-resolution scanning electron microscopy (SEM). These mitochondria formed a continuous network. On the basis of their morphological characteristics, the mitochondrial networks were roughly classified into four types. Type-1: Mitochondrial networks are composed of a single branched tubule of almost uniform thickness of about 250-300 nm. Type-2: Mitochondrial networks have two kinds of tubules, about 250-300 nm and 100 nm across. Type-3: Mitochondrial networks are composed of two different parts; globular parts around 1.0 μm in diameter and filamentous parts about 50-100 nm across. Type 4: Highly complex mitochondrial networks consisting of multiple parts of varying shapes. The significance of this morphological diversity of mitochondrial networks is discussed with reference to recent studies involving other techniques, notably light- and transmission electron microscopy while considering the advantages and disadvantages of the SEM methods employed in this study.
Fluorescence microscopy is a common tool in the field of the life science for specific labeling and observation of target molecules. Methods of non-invasive staining have been established by ectopic expression of fluorescence proteins, with which the fluorescence microscopy has been applied to live-cell imaging. Superresolution microscopy circumventing the diffraction limit of light has also been developed recently. Here we focus on one of the superresolution microscopic modalities, photoactivated localization microscopy (PALM). For PALM, a target molecule is labeled with a photoswitchable fluorescent protein. Using a stochastic switching of the fluorescent protein, spatially overlapping molecules can be temporally isolated, and the coordinates of respective molecules are calculated from fitting of the signals to the two-dimensional Gaussian function. With PALM, the target molecules can be observed with the resolution of tens of nm. By staining cells with recently developed probes, PALM is also applicable for the imaging of membrane rafts. It can also be used for the single particle tracking in living cells to analyze molecular dynamics.
Morphological observation of organelle in the mature rice seed is poorly understanding, because the electron microscopy of mature rice seed is technically difficult. We attempted to modify the existing method of embedding rice grain in resin. Modified method revealed the ultrastructures of mature organelle in dry rice seed. Observation of subcelluler structures in mature starchy endosperm in rice grains will be useful in future to farmers, breeder, consumers and food processors. Moreover, this method should provide a highly useful tool for observation of organelle and understanding seed development of other cereals.
The features and capabilities of depth-resolution imaging by aberration-corrected transmission electron microscopy (TEM) are explained in terms of phase-contrast transfer functions and Fresnel propagation of electron wave fields. The depth-resolution imaging is useful for obtaining three-dimensional information of nano structures as shown in the successful examples; detection of the inclination angle of a carbon nanotube (CNT), visualization of the grade separation crossing of CNTs, and thickness determination of an amorphous carbon film supporting a gold nano particle. In addition, a new standard for the exact focal plane is discussed in order to treat a finite-thickness material as a phase object. Based on the discussion, the depth-selective imaging is explained referring to the successful examples, in which upper and lower side wall lattices of a single-wall CNT were separately observed and a medium-range-order structure embedded in Zr66.7Ni33.3 metallic glass was detected using aberration-corrected TEM.
Neurons form a three-dimensional network to constitute neuronal circuits that are responsible for a wide variety of brain functions. The functional mechanisms of the brain can therefore be elucidated by analyzing the three-dimensional structure of brain tissue. We have revealed brain tissue structures by synchrotron-radiation X-ray microtomography and determined neuronal circuits in them. In this paper, we illustrate the technical aspects of the microtomographic analysis and report examples of neuronal network structures.
Strain measurement using a moiré fringe in scanning transmission electron microscopy (STEM) is reported. The moiré fringe in STEM arises as an interference fringe of a crystal lattice and a scanning raster, due to under-sampling effect. The moiré spacing is magnified with the factor determined by the ratio of the raster spacing with respect to crystal lattice spacing, when they are parallel. The width of the fringe is controllable, since we can arbitrarily select the spacing and direction of the raster. The strain map of the crystal is calculated with the phase shift of the moiré fringe. And the calculation is based on a phase analysis procedure developed for holography. The specimen drift compensation, using a non-strained reference region, was applied to improve the final accuracy. And the averaging of resulted multiple phase maps derived from moiré patterns was also applied to the final results to improve the accuracy. The accuracy of the strain map, examined with a non-strained Si crystal, was approximately 0.2 % in standard deviation of the strain map with a spatial resolution of several nanometers.