SEM observation of resin-embedded sections is attracting attention as a method to observe wide area of an organ cross section and also to grasp 3D structure of it by observing serially sectioned samples. In the section observation, not only observing conditions such as selection of the detector or acceleration voltage but also sample pretreatment factors such as thickness of the section or choice of the substrate are complicatedly related to each other. It is important to understand characteristics of the SEM to use and to select appropriate pretreatments and observing conditions. This article explains the differences made by selection of acceleration voltage, sample thickness, and pretreatments in backscattered electron images mainly used in SEM observation of sections.
The scanning electron microscope (SEM) has often been used in plant research to observe the surface structure of organs and tissues such as leaves and roots. Technological innovations developed in recent years, such as electron guns and detectors for SEM, have made it possible to take electron microscopic images of tissues, organelles, and cells in sections that are equal in quality to those from transmission electron microscopy (TEM). To achieve this quality, resin-embedded sections containing biological samples are placed on a flat plate, such as a glass slide or cover slip, and then observed with a SEM using a low acceleration voltage beam and a backscattered electron (BSE) detector. Thereby, wide-area electron microscopic images can be easily obtained using sections thicker than a TEM section. In this article, we reported effective imaging conditions using a field emission SEM equipped with an yttrium aluminum garnet-BSE detector, and applied it to ultrastructural analyses by wide-region observation using resin-embedded sections of plant samples. By using serial sections, a three-dimensional reconstruction was achievable. In addition, we described the results obtained by correlative light and electron microscopy using this technique.
Biological tissues have extremely fine and complex morphological characteristics, and currently, electron microscopy (EM) is indispensable for observing and imaging these ultra-microstructures. However, the biological tissue imaging setup in conventional EM analysis limits the area of observation. Thus, it is challenging to perform a comprehensive and quantitative morphological analysis of the whole tissue using EM. In this section, we have introduced a new method called the “large-area section SEM method,” which enables continuous imaging of huge tissue sections at a resolution of EM level with a high speed over a wide area. The latest scanning-type EM technology equipped with automatic imaging technology was used to observe conventional resin-embedded tissue sections. In addition, a wide-area stereoscopic observation method using array tomography and a quantitative analysis method for EM images using deep learning have been outlined in this section. We look forward to establishing a method to analyze large biological tissue imaging data, the so-called comprehensive morphological analysis “morphomics analysis.”
Section scanning electron microscopy (SEM) is a novel SEM technique based on observation of backscattered electron (BSE) images of tissue sections embedded in resin on glass microscope slides. Using this technique, high-resolution transmission electron microscopy (TEM)-like images can be obtained without the need to use specialized techniques such as ultramicrotomy. In this paper, we introduce three techniques based on the SEM method: 1) BSE imaging of semithin sections; 2) correlation of immunofluorescence and BSE images of the semithin sections; and 3) combination of a cryosectioning method (the Tokuyasu method) with section SEM. Additionally, we describe serial section SEM, which is a novel 3D imaging method, and discuss the possibilities for use of these SEM imaging techniques for tissue sections.
A magnetic skyrmion is a nanometer-scale vortex-like magnetic structure stabilized in magnets without spatial inversion symmetry. The magnetic skyrmions have been actively investigated for their possible applications to the next generation spintronics devices. For their practical applications, understanding their structures and their response are indispensable. Lorentz transmission electron microscopy is an effective method for observing magnetic skyrmions directly in real-space. Lorentz transmission electron microscopy has been playing an important role in research of magnetic skyrmions combined with other observation and analytical techniques of transmission electron microscope. Here, we review some researches on magnetic skyrmions by techniques of transmission electron microscopy and data analysis.
An atomic force microscope (AFM) enables to describe the surface structure by tracing the sample surface with a probe (needle) even in water. Its resolution is almost equivalent to that of an electron microscope. Therefore, it is expected that the motility of cell surfaces can be viewed at living state in culture medium. However, in practice, we had to wait for the development of today’s high-speed AFM, because the probe had to scan several micron squares faster than the movement of the cell surface for detecting the surface structure in detail. The AFM used (Olympus BIXAM) captures an area of 6 μm × 4 μm at a rate of 1 frame/10 seconds. Although it is not necessarily fast, the movements of lamellipodia extending from the cell surface and actin filaments beneath the cell membrane were clearly detected. On the other hand, application of unroofing method and cryo-sectioning method to AFM sample preparation made it possible for the first time to observe the intracellular structure in water by AFM.
Recently, it has been demonstrated that single particle analysis using 200 kV electron cryo-microscope is capable of reconstructing protein structures at resolution higher than 3.0 Å. Because of the enhanced contrast of particle images taken by 200 kV microscope compared with 300 kV, proteins smaller than 100 kDa were also reconstructed at the same resolution level without using large defocus or phase plate. This is, the 200 kV should be more suitable for small proteins (< 200 kDa). However, the majority of near-atomic resolution cryo-EM structures has been determined using 300 kV. As a consequence, many of typical parameter settings for imaging session and image processing steps, especially ones associated with the contrast transfer function, are based on the accumulated experience of the higher acceleration voltage. Here, we will revise these parameters for 200 kV, establish theoretical base for criteria to find an optimal box size and particle mask diameter for a given dataset, and explain a proposed protocol. Examples of actual analysis are also given. In these, by considering the defocus distributions, merely optimizing the box sizes and particle mask diameters yielded prominent improvements from resolutions around 3.0 Å in the reconstructions of < 200 kDa proteins.