Microscopy cryomicroscopy

A completely new method of determining siufaces arises from the enormous developments in electron microscopy. In contrast to the above-mentioned methods where the surfaces were calculated, molecular surfaces can be determined experimentally through new technologies such as electron cryomicroscopy [188]. Here, the molecular surface is limited by the resolution of the experimental instruments. Current methods can reach resolutions down to about 10 A, which allows the visualization of protein structures and secondary structure elements [189]. The advantage of this method is that it can be apphed to derive molecular structures of maaomolecules in the native state. 

Electron cryomicroscopy, 2, 94-101, 401-2 field depth/resolution graph for, 100 instrument choices in, 97-101 theoretical consideration of, 94-101 three dimensional reconstruction and, 101 Electron density maps, 42 Electron microscopy resolution (EM resolution), 45-46... 

Many medium resolution structures of macromolecular assemblies (e.g., ribosomes), spherical and helical viruses, and larger protein molecules have now been determined by electron cryomicroscopy in ice. Four atomic resolution structures have been obtained by electron cryomicroscopy of thin 2D crystals embedded in glucose, trehalose, or tannic acid (11-14), where specimen cooling reduced the effect of radiation damage. One of these, the structure of bacteriorhodopsin (1 l)provided the first structure of a seven-helix membrane protein. The medium resolution density distributions can often be interpreted in terms of the chemistry of the structure if a high resolution model of one or more of the component pieces has already been obtained by X-ray, electron microscopy, or NMR methods. As a result, the use of electron microscopy is becoming a powerful technique for which, in some cases, no alternative approach is possible. Useful reviews [e.g., Dubochet et al. (9), Amos et al. (15), Walz and Grigorieff (16), and Baker et al. (17)] and a book [Frank (18)] have been written. 

Fig. 7. Side view of the EcFo F, (left) and CFo F, (right) ATP synthases obtained by cryoelectron microscopy. See text for discussion. Figure source (A) Capaldi, Aggeler, Turina and Wilkens (1994) Coupling between catalytic sites and the proton channel in F F -type APTases. Trends in Biochem Sci 10 285 (B) Bottcher, LUcken and GrSber (1995) The structure of H -ATPase from chloroplasts by electron cryomicroscopy. Biochem Soc Trans 23 783. (B) is kindly provided by Dr. Peter Graber.

These new methods, low-vacuum, cryomicroscopy and environmental microscopy, minimise damage to the delicate hydrate in cementitious materials (notably C-S-H). However, as illustrated in Section 8.3, the differences in morphology compared to carefully prepared conventional samples are not dramatic and support the view that conventional SEM can give images... 

---