ABSTRACT
This chapter summarizes the preparation of nanomembrane and their subsequent characterization by transmission electron microscopy (TEM) utilizing new support materials such as metallic TiSi glasses, doped silicon carbide, and two-dimensional carbon materials such as carbon nanotube and grapheme. 6.1 Introduction: Transmission Electron
Microscopy of Biological SpecimensKnowledge about the structure of proteins and macromolecular complexes is indispensable for understanding biological processes on the molecular level. The structure of biomolecules can be determined by X-ray crystallography, nuclear magnetic resonance
(NMR) spectroscopy, and TEM. Transmission electron microscopy is a powerful tool for structural biology as it covers the entire range of biological structures ranging from single molecules up to tomographic volumes of whole cells [1-3]. Moreover, TEM enables the structural analysis of non-periodic biological structures, whereas X-ray crystallography requires large, well-ordered three-dimensional crystals of biomolecules.To preserve the structure of biomolecules in a near-native state, they are vitrified in a thin water layer by shock freezing in liquid ethane and analyzed at the temperature of liquid nitrogen or liquid helium. Cooling the specimen reduces the impact of radiation damage [4-6]. The goal of single-particle electron cryo-microscopy (cryoEM) is to unravel the structure of biomolecules at atomic or near-atomic resolution. Ultimately, the amino acid chain of a protein can be fitted into an electron density map obtained by TEM. The structure of several vitrified biomolecules has been solved at atomic or near-atomic resolution by cryoEM from two-dimensional crystals [7-11], helical arrays [12,13], and as single particles [14-19]. 6.1.1 Electron Cryo-Microscopy of Ice-Embedded
Many successful efforts have been made to improve instrumentation for TEM. Energy filters remove inelastically scattered electrons [20], correctors compensate for spherical and chromatic aberration [21,22], and physical phase plates are used to generate in-focus contrast for biological specimens [23-30], and CMOS detectors enable data acquisition with unprecedented signal-to-noise ratio [17,31,32]. Noteworthy, the resolution of modern aberrationcorrected TEMs exceeds 0.5 Å in materials science applications [33]. Although theory predicts that atomic resolution should be routinely achieved with vitrified biomolecules [34], resolutions obtained with most ice-embedded specimens are considerably worse. The most critical factor in biological high-resolution cryoEM is the specimen itself.For single-particle cryoEM, biomolecules are embedded in a thin layer of vitreous ice spanning the holes of a holey carbon film (Fig. 6.1).
