Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo‐sectioning approaches, as a comprehensive toolset enabling high‐resolution direct cryo‐TEM observation of a broad range of LCs. We also present comparative analysis using cryo‐TEM and replica freeze‐fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high‐pressure freezing and cryo‐sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo‐TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent‐core thermotropic LCs, comprehensive understanding of the twist‐bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754–772, 2014. © 2014 Wiley Periodicals, Inc.

K E Y w O R D s. High-pressure freezing, cryosectioning, cutting artefacts, plant leaves, diamond knives, ionization electrode, low-temperature microscopy, energy filter.High-pressure frozen Golden Delicious apple leaves were cryosectioned at low temperature with diamond knives. Good cryosections were obtained by optimizing the cutting parameters, i.e. sectioning temperature, mechanical stability of the sample, and sectioning velocity. Cutting artefacts were minimized by reducing the electrostatic interactions between the knife surface and the cryosection. This was accomplished by sectioning the sample in the presence of an ionization electrode. The ionization device, with a primary voltage of 7-8 kV, produces positively and negatively charged nitrogen ions which neutralize the surface charges of the knife and the section. This minimizes the friction on the knife surface and results in ultrathin sections without crevasses or knife marks. Compression of the sections could be minimized, but not avoided, by reducing the knife angle to 30". Improved contrast of the frozen-hydrated sections was obtained with the Zeiss E M 902 energy-filter microscope operated in the zero-loss mode.

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Biological hard tissues are a rich source of design concepts for the generation of advanced materials. They represent the most important library of information on the evolution of life and its environmental conditions. Organisms produce soft and hard tissues in a bottom-up process, a construction principle that is intrinsic to biologically secreted materials. This process emerged early on in the geological record, with the onset of biological mineralization. The phylum Brachiopoda is a marine animal group that has an excellent and continuous fossil record from the early Cambrian to the Recent. Throughout this time interval, the Brachiopoda secreted phosphate and carbonate shells and populated many and highly diverse marine habitats. This required great flexibility in the adaptation of soft and hard tissues to the different marine environments and living conditions. This review presents, juxtaposes and discusses the main modes of mineral and biopolymer organization in Recent, carbonate shell-producing, brachiopods. We describe shell tissue characteristics for taxa of the orders Rhynchonellida, Terebratulida, Thecideida and Craniida. We highlight modes of calcite and organic matrix assembly at the macro-, micro-, and nano-scales based on results obtained by Electron Backscatter Diffraction, Atomic Force Microscopy, Field Emission Scanning Electron Microscopy and Scanning Transmission Electron Microscopy. We show variation in composite hard tissue organization for taxa with different lifestyles, visualize nanometer-scale calcite assemblies for rhynchonellide and terebratulide fibers, highlight thecideide shell microstructure, texture and chemistry characteristics, and discuss the feasibility to use thecideide shells as archives of proxies for paleoenvironment and paleoclimate reconstructions.

SUMMARY Electron microscopy of vitrified ultrathin sections allows cell ultrastructure to be studied in the hydrated state. Sectioning of the frozen material is, however, a limiting step, since the cutting forces cause severe mechanical deformation. In order to address this problem, we have investigated the surface of cryosections. It is shown that cryosections have two fundamentally different surfaces. One surface is rough, deformed by cutting‐induced deformation lines which are orientated perpendicular to the cutting direction. The other surface, in comparison, is not affected by those deformation lines. Except for knife marks it is smooth. In order to explain the observations, the following model is proposed. The rough relief corresponds to the former block face. Its roughness originates from material that is squeezed out of the section plane when the section is compressed in the cutting direction and bent away from the specimen block. The smooth section surface is the surface in contact with the knife during the sectioning. This contact keeps the surface smooth while imprinting the knife marks.