Soft materials such as polymers and biological samples often consist of light elements. A commonly encountered problem in transmission electron microscopy (TEM) studies is the low intrinsic contrast between constituent species due to the slight difference in the electron density of different organics or polymers. In this case, selective staining of one of several components with reactive species containing heavy atoms, i.e., the staining agents, is necessary to visualize the morphology. The representative staining agents are osmium tetroxide (OsO₄) and ruthenium tetroxide (RuO₄). Each staining agent uses a reaction unique to its structure, e.g., OsO₄ selectively stains double bonds by forming a complex. Various precautions must be taken in handling the staining agents because of their toxic nature. They must be safely stored, used in the exhaust system. Moreover, various artifacts, e.g., over staining, under staining, contamination and depth dependence during staining, could affect accurate determination and quantification of structures. This article describes how to deal with the above staining-related problems for better observations of polymeric and biological samples.

In order to observe the microstructure of soft materials with an electron microscope, it is necessary to thin the specimen. In many cases, ultramicrotomy is used for specimen preparation to ultra-thin section or cross-sectional samples. For analysis of central part of bulk specimen, it can be observed with ultra-thin sections prepared by ultramicrotomy without embedding. On the other hand, to make ultra-thin sections such as the surface of bulk specimen, films, and fibers, it is necessary to embed them with resin. For embedding soft materials with resin, the choice of resin is very important, because in the case of improper resin, “artifacts”, which are completely different from the original structure, are formed. When embedding soft materials, it is ideal “not too strong adhesion” between the specimen and the resin, therefore it needs to consider the solubility parameter of the specimen and the resin. Also, if the thin sectioning with ultramicrotome is not appropriate, “artifacts” are formed too. This article provides examples of artifacts resulting from the embedding or the thin sectioning and describes how to avoid them.

Electron beam irradiation induces various damages to specimens, and most embedding resins are known to shrink by several tens of percent during the irradiation. The detailed mechanism of this shrinkage has not been clear, but it has been shown that the ultrathin sections on a grid shrink at a same rate within the section mainly in the direction perpendicular to the grid and little in the horizontal direction. The shrinkage tends to occur rapidly at the beginning of beam irradiation and gradually stabilize, and the process can be approximated by a logarithmic function of the electron dose. Cryo-temperature can suppress this deformation only at low dose irradiation. Understanding the deformation characteristics of the resin during beam irradiation is extremely important for image interpretation in electron microscopy of soft materials and for optimizing the pre-irradiation dose in electron tomography.

Several applications of ion beam SEM/TEM sample preparation for soft materials such as organic polymer are shown. Although conventional ultramicrotomy is useful tool for sample preparation for soft matter, it is hard to cut the specimen in the case of including inorganic hard materials such as CFRP. Ion beam milling such as BIB or FIB can avoid physical damage, however by using conventional strong ion beam such as Ga+, soft materials are easily degraded by knock-on and heating damage processes. We show how to overcome these ion-damage problems by electro-staining and cryo-technique. In addition, we also show that plasma FIB can provide wide and flat cross section for organic/inorganic complex such as CFRP.

This article will discuss the resolution, one of the essential performance indicators in scanning electron microscopy (SEM). The former part outlines definitions of resolution used in SEM and its evaluation methods. It also describes the image sharpness evaluation developed for the international standardization of image resolution. The second half discusses the importance of specimens in assessing the resolution of SEM images and introduces the certified reference materials for image sharpness evaluation provided by the National Metrology Institute of Japan.

In this decade, electron microscopies (EM) specialized for acquisition of serial electron micrographs of the brain tissue and the deep learning based automated dense segmentation of the neural elements in the volume have been developed efficiently. Now, it is possible to take one cubic millimeter volume of the brain tissue EM data set in a high magnification capable of identifying synaptic connections, and connectome of the brain tissue would be feasible with the 3D reconstructions obtained with the dense segmentations. Here, the outline of the electron microscopies for the large volume EM data set acquisition and its outstanding technologies are briefly introduced.

A combination of scanning tunneling microscopy with plasmonics allows us to perform ultrasensitive and ultrahigh resolution optical spectroscopy down to the atomic/molecular scale. I introduce recent research and techniques that has been developed by the author’s group.

The combination of cryo-SEM and rapid freezing has enabled us to observe biological samples without chemical fixation. It is also possible to observe emulsion, slurry and gels. Although cryo-SEM technology is really useful, it is less well known. This article describes the Cryo-SEM procedures and required equipment for a wide range of “Kenbikyo” readers.