168 5.2  Electron Microscopy

involving no dehydration step at which the sample temperature throughout, not just the fix­

ation step but the entirety of the investigation from sample preparation through to the final

imaging acquisition, has been kept below 140 K, which is the vitrification temperature of

water, or in other words biological samples with very minimal sample preparation artifacts.

These investigations require a specialized cold stage, typically using liquid nitrogen (boiling

point 77 K, which allows a stable cold stage of 110 K to be maintained) or, in some advanced

machines, liquid helium (boiling point 4 K).

Cryo-​EM is particularly useful as a structural biology tool, both using metallic shadowing

and negative staining techniques, and can be applied in transmission and scanning modes.

For molecular-​level structural investigations, cryo-​EM is used for superior spatial reso­

lution compared to SEM. However, the absolute level of spatial resolution in raw cryo-​TEM

molecular reconstructions is still an order of magnitude worse than the definitive atomic-​

level resolution achievable by the techniques of nuclear magnetic resonance (NMR) and x-​

ray crystallography. However, improvements in the methods of image analysis in particular

mean that cryo-​EM in many cases rivals the traditional atomic-​level structural biology

methods.

For example, the inferior raw spatial resolution of EM compared to the atomistic-​level

structural biology techniques can be improved by subclass averaging. This operates by cat­

egorizing each raw image of a molecular complex into a distinct class of image type, aligning

each image within that class and then generating a single average image for each subclass.

In the early days of this technique, in fact, close to the turn of the twentieth century, such

averaging was performed manually, in a highly precarious and potentially subjective way.

However, improvements in modern subclass categorization methods involve principal com­

ponent analysis of eigenimages (originally described as eigenfaces from its implementation

in face recognition software), although there are still potential issues with user-​defined

thresholds for determining and recognizing subclass features (discussed in Chapter 8).

However, cryo-​EM also has some important advantages over x-​ray crystallography

and NMR, in that it can be applied to molecular complexes that are >250 kDa in summed

molecular weight, which is far greater than NMR (~90 kDa maximum) and can be applied

to intact large molecular complexes unlike x-​ray crystallography, which requires the forma­

tion of highly pure crystals, which are too difficult to generate either because they require

the presence of a phospholipid bilayer to form stably or because they consist of multiple

molecular components. These include not only the large membrane complexes of the fla­

gellar motor and ATP synthase mentioned earlier but also certain essential macromolecular

complexes in the cytoplasm such as the intact ribosome and large intact viruses. The ribo­

some is a particularly good example since the separate components of a ribosome can be

purified and structures are determined by x-​ray crystallography, whereas to visualize the

entirety intact ribosome requires a technique such as cryo-​EM.

Electron cryotomography (CryoET) is a specific application of cryo-​EM for which 3D

images can be reconstructed from multiple 2D images of a sample obtained by tilting over

a range of orientations up to a limit of around 70˚. Since the electron propagation distance

though the sample increases during tilting, this imposes a practical sample thickness upper

limit to avoid significant electron beam attenuation, typically around 0.5 µm. Many CryoET

studies to date have thus focused on unicellular microbes and viruses, and macromolecular

complexes, though thinning of larger samples can be performed using focused ion beam (FIB)

milling, in addition to normal cryo-​sectioning. CryoET may also be combined with fluores­

cence microscopy methods to generate more specificity for identifying cellular structures,

using similar correlative approaches to those described in Section 5.2.7.

5.2.7  CORRELATIVE LIGHT AND ELECTRON MICROSCOPY

Correlative light and electron microscopy (CLEM) combines the advantages of the time-​

resolved fluorescence microscopy on live cellular material with the higher spatial reso­

lution achievable with EM. As we discussed in Chapter 4, fluorescence microscopy offers

a minimally invasive high-​contrast tool, which can be used on live-​cell samples to monitor