Electron microscopy of biological specimens in liquid water.

Mirsaidov et al. (1xImaging protein structure in water at 2.7 nm resolution by transmission electron microscopy. Mirsaidov, U.M., Zheng, H...., and Matsudaira, P. Biophys. J. 2012; 102: L15–L17Abstract | Full Text | Full Text PDF | PubMed | Scopus (36)See all References1) have used a microfabricated, thin-window wet cell to examine aqueous biological specimens in the electron microscope. While the thin-window technology used in their work represents a significant innovation, there is no reason to expect that the fundamental consequences of radiation damage could be changed by this technology.As the authors point out, earlier work had led to the conclusion that protein structures are damaged more rapidly by exposure to the electron beam when in liquid water than when in the frozen-hydrated state. The increase in tolerable radiation exposure that is provided by freezing protein crystals has subsequently been confirmed in the context of x-ray crystallography, as is reviewed in Nave and Garman (2xTowards an understanding of radiation damage in cryocooled macromolecular crystals. Nave, C. and Garman, E.F. J. Synchrotron Radiat. 2005; 12: 257–260Crossref | PubMed | Scopus (72)See all References2) and Garman and Weik (3xMacromolecular crystallography radiation damage research: what's new?. Garman, E.F. and Weik, M. J. Synchrotron Radiat. 2011; 18: 313–317Crossref | PubMed | Scopus (9)See all References3). Nevertheless, Mirsaidov et al. (1xImaging protein structure in water at 2.7 nm resolution by transmission electron microscopy. Mirsaidov, U.M., Zheng, H...., and Matsudaira, P. Biophys. J. 2012; 102: L15–L17Abstract | Full Text | Full Text PDF | PubMed | Scopus (36)See all References1) now claim that the contrary is true for a crystalline bundle of actin filaments. Mirsaidov et al. also suggest that radiation damage might be less in a liquid environment than in the solid (frozen-hydrated) state because radicals could diffuse away from a protein rather than stay and do further damage. This suggestion is contrary to current thinking, however, in which caging of radiolysis products, in the solid state, is thought to be important in retaining a close-to-original localization of atoms. Furthermore, it is believed that low-temperature immobilization of radicals produced in the surrounding solvent reduces their diffusion toward the protein, thereby reducing the contribution that they make to secondary (chemical) damage of the protein.Perhaps there are other, more plausible alternatives that correctly explain the measurements reported by Mirsaidov et al. If, for example, their specimens had inadvertently become air-dried within the wet cell, the specimens could have become embedded in buffer, salt, or other nonvolatile solutes, resulting in negatively stained specimens such as those described by Massover and co-worker (4xUnconventional negative stains: Heavy metals are not required for negative staining. Massover, W.H. and Marsh, P. Ultramicroscopy. 1997; 69: 139–150Crossref | Scopus (15)See all References, 5xOn the experimental use of light metal salts for negative staining. Massover, W.H. Microsc. Microanal. 2008; 14: 126–137Crossref | PubMed | Scopus (12)See all References). In this case, it would not be surprising that the radiation tolerance of their sample would exceed that of frozen-hydrated specimens. Indeed, the image shown in Fig. S1 C in the Supporting Material looks similar to that of a negatively stained specimen, the objects in Fig. 1, B and F, could be interpreted as being air-dried samples, and specimen drying during irradiation (albeit at much higher electron exposures) is described in the last paragraph of the Supporting Material. It thus would have been informative if the authors had extended the measurements shown in Fig. 2 to determine whether diffraction spots at a resolution of ∼5 nm actually last indefinitely, as they generally do for negatively stained specimens (6xLimitations to significant information in biological electron microscopy as a result of radiation damage. Glaeser, R.M. J. Ultrastruct. Res. 1971; 36: 466–482Crossref | PubMed | Scopus (144)See all References6). A higher standard of proof is thus needed to convincingly make the claim that structural damage occurs less rapidly in liquid water than in ice. For example, these studies should be extended to include thin catalase crystals in order to make a direct comparison to earlier work with wet-hydrated and frozen-hydrated samples.The authors emphasize the goal of using wet specimens (as opposed to using frozen-hydrated, glucose-embedded, or other solute-embedded specimens) to observe protein dynamics with nanometer resolution. A point that needs to be considered, however, is that enzymatic function is much more sensitive to radiation damage than is overall protein structure (at the nanometer level of resolution). For example, very few proteins remain enzymatically active after receiving radiation doses of 100 Mrad (1 MGy) (7xSize determination of enzymes by radiation inactivation. Kempner, E.S. and Schlegel, W. Anal. Biochem. 1979; 92: 2–10Crossref | PubMed | Scopus (137)See all References7), whereas structural features can tolerate—in frozen-hydrated specimens—up to 100 times that dose, depending upon the resolution. Note that 100 Mrad =1010 erg deposited per gram, and 1 gray (Gy) = 100 rad. Because the energy deposited per gram of protein is ∼60 Mrad (0.6 MGy) when a single frame is recorded with an exposure of 10 electron/nm2 (for 200 keV electrons), a case must be made that biological specimens can, at least in theory, remain active over the course of recording two or more images at nanometer resolution.