An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology

Abstract

Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins >150kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a 'resolution revolution', owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM.

Keywords: Electron microscopy.

Fig. 1

Example workflow for structure determination by single particle EM.

Fig. 2

Positive and negative staining using heavy metal salts. In negative staining (A) the stain fully envelops the macromolecular complex; in the micrograph the complex appears white on a dark background. Positive staining (B) results in a small amount of stain forming a thin shell around the molecule, meaning the sample appears dark against a light background.

Fig. 3

Examples of vitreous and non-vitreous ice. A) Empty, vitreous ice (Scale bar 50 nm). B) Hexagonal ice (scale bar 400 nm). C) Large ice crystal (white arrow) (scale bar 400 nm). D) Probable ethane contamination (scale bar 200 nm).

Fig. 4

Multi-scale imaging by cryo-EM. A) Eukaryotic cells (scale bar 6 μm). B) Prokaryotic cells (scale bar 0.5 μm). C) Isolated organelles, in this case microsomes (scale bar 200 nm). D) Synthetic liposomes (scale bar 100 nm) E) Viruses (scale bar 50 nm). F) Macromolecular complexes (scale bar 25 nm).

Fig. 5

Examples of EM support films. A) Holey carbon, such as Quantifoil^® R2/2 (2 μm holes separated by 2 μm), Scale bar 1 μm. B) Lacy carbon film (irregular network of thin carbon), Scale bar 1 μm. C) Finder grid (here with Quantifoil^® R2/2 carbon), scale bar 10 μm.

Fig. 6

Number of structures in the EMDB. A) Cumulative map releases in the EMDB between 2002 and 2015. B) Map releases at given resolution levels between 2002 and 2015.