Cryo-temperature stages in nanostructural research

Abstract

Cryo high-resolution scanning electron microscopy (cryo-HRSEM) also called low temperature (LT)-HRSEM, is the most powerful tool for visualizing 3D topographic ultrastuctural features of solid-state solvated systems. A cryostage provides researchers with the ability to image their cryoimmobilized solvated system, usually aqueous, and image them in the solid-state at cold temperatures with high magnification (5 × 104 to 106), such that structural information down to a single nanometer can be obtained. LT-HRSEM is particularly a useful imaging mode for soft materials such as cells, biomolecules, biomaterials, and organic systems. Hydrogels, colloids, suspensions, emulsions, and whole cells all have ultrastructural components that can be imaged on a nanometer scale when the specimens have been LT cooled and transferred onto a cryostage, and then into the HRSEM. Numerous advantages result from low-temperature imaging provided that the sample is prepared as small as possible and kept frost-free. Additionally, cryo-HRSEM is ideally suited for imaging solid structures in nonaqueous or organic solvents such as toluene and TFE, or in organic fluids such as octanol. Low-temperature "bulk" specimens are prepared for cryo-HRSEM in microliter volume sample sizes. Such samples, in both nonaqueous and aqueous systems, provide vast vistas for acquisition of nanometer size ultrastructural information at high magnification. For instance, a representative aqueous aliquot is prepared that can contain thousands of cells or biomolecular complexes. LTHRSEM images of nanoparticles can be obtained within the context of complex biological compartments. There is no need to prepare larger samples because the greater mass of a larger specimen will serve only to reduce the cooling rate and result in unwanted crystallized systems. Since secondary electron microscopy is a surface imaging electron optical mode, relief of the solid components in a bulk LT aqueous sample is sometimes desirable or necessary. Solid water may be removed by high vacuum (10-7 Torr) sublimation. This method is referred to as "etching." A detailed discussion of low temperature preparation follows but the reader should realize that practitioners of LT-HRSEM take great efforts to remove the bulk LT aqueous phase without removal of the hydration shell surrounding molecular structures observed in the nanometer range. Removal of molecular hydration shell is the process of freezedrying, which is not the aim of LT-HRSEM on bulk specimens. Alternatively, raising the temperature to-85°C under vacuum of 10-7 Torr to freeze-dry LT samples has been successful for 1-10 nm structural resolution of thin biomolecular specimens mounted on grids and then imaged by LT-HRSEM [1,2]. It is precisely the HRSEM's ability to image surfaces with topographic contrast on relatively large bulk LT samples, as compared to small cryo-TEM samples, that facilitates its expanded applications in nanostructural studies. Cryo-HRSEM, just as with the cryo-TEM, can be used to examine single isolated biomolecules and macromolecular complexes (single particle analysis), which are considered as "thin" specimens. Thin specimens are cryoimmobilized by vitrification in a 100 nm thick aqueous layer on carbon-coated grids. The topic of vitrification will be covered in the section dealing with cryoterminology. Conveniently, a thin vitrified specimen can be cryo transferred into a SEM fitted with a scanning transmission electron microscopy (STEM) detector and tandem cryo-HRSEM/cryo-STEM can be performed. It is now appropriate to provide definitions used in cryogenic preparation and imaging. © 2006 Springer Science+Business Media, LLC.