53] Electron microscopy of frozen hydrated specimens: Preparation and characteristics

Abstract

Publisher Summary This chapter describes the preparation and characteristics of the electron microscopy of frozen hydrated specimens. Frozen hydrated biological specimens are observed in the electron microscope. Quantitative structural analysis seems to show that no artifact is introduced during freezing and that the biological entity has a similar structure in the solid amorphous aqueous state as in the liquid aqueous state. The main equipment for observing frozen hydrated specimens is an electron microscope equipped with a mechanically and thermally stable cold specimen holder. The preparation of frozen hydrated specimens involves thin aqueous layer preparation and the cooling of the thin aqueous layer. Structural preservation of biological objects, freezing rate, and the forms of ice are also described in the chapter. It discusses the characterization of frozen hydrated specimens on the basis of structural preservation, beam damage, the contrast and hydration of unstained, frozen specimens, and high-resolution imaging of unstained, frozen-hydrated specimens. Cryospecimens are fully hydrated when they are embedded in amorphous solid water. Vitrified specimens are mainly visualized by phase contrast and need to be imaged with defocus values.