Chapter 5 – Detection and Imaging Tools that Use Nonoptical Waves  165

5.2.3  GENERATING CONTRAST IN ELECTRON MICROSCOPY

Biological matter is mostly water and carbon, comprising relatively low-​atomic-​number elem­

ents. This results in a far greater mean free collision path for electrons in carbon compared

to high-​atomic-​number metals. For example, at a 100 kV accelerating voltage, an electron in

carbon has a mean free collision path of ~150 nm, whereas in gold, it is ~5 nm. To visualize

biological material therefore, a high-​atomic-​number metal contrast reagent is applied.

Negative staining can be applied on both chemically and cryofixed samples, usually by

including a heavy metal contrast reagent such as osmium tetroxide or uranyl acetate dissolved

in an organic solvent such as acetone. The contrast reagent preferentially fills the most access­

ible volumes in the sample (those least occupied by the densest biological matter). This there­

fore results in a negative image of the sample if the electrons are transmitted onto a suitable

detector. This technique can generate excellent contrast between heterogeneous biological

matter found in vivo (e.g., illustrated in the case of muscle tissue in Figure 5.1b).

Another contrast reagent incorporation method involves positive staining via metallic

shadowing, typically of evaporated platinum. This not only can be applied to relatively large

length scale samples (e.g., small whole organisms such as insects) to coat the surface for visu­

alization of backscattered electrons reflected from the metallic coat but is also a common

approach applied to visualizing single molecules from visualization of transmitted electrons

through the sample. Here, a dilute purified solution of the biomolecules is first sprayed onto

a thin sheet of evaporated carbon, which is supported from an EM-​grid sample holder. The

sample aqueous medium is then dried in a vacuum and platinum is evaporated onto the

sample from a low angle <10° as the sample is rotated laterally.

This creates a uniform metallic shadow of topographical features of any single molecules

stuck to the surface of the carbon, which are electron dense, generating a high scatter signal

from an electron beam, whereas the supporting thin carbon sheet is relatively transparent to

electrons and thus results in a “positive” image. Single gold or platinum atoms have a diam­

eter of ~0.3–​0.4 nm, but typically, a minimum-​sized cluster of atoms in a shadowed single

region in a sample might consist of 5–​10 such atoms, in which case the real spatial resolution

may be worse than expected, after the effects of diffraction and spherical aberration, by a

factor of ~2–​4.

Metallic shadowing is also used for generating contrast in freeze-​fracture samples. Here,

larger angles of ~45° are applied in order to reach more recessed surface features compared

to single-​molecule samples. This method can generate excellent images of the phospholipid

bilayer architecture of cell membranes, down to a precision sufficient to visualize single polar

head groups of a phospholipid molecule.

Both tissue-​/​cellular-​level samples and single-​molecule samples can also be visualized

using immunostaining techniques. These involve incubating the sample with a specific anti­

body, which contains a heavy metal tag of just a few gold atoms. The antibodies will then bind

with high affinity to specific molecular features of the sample, thus generating high electron

beam attenuation contrast for those regions, often used to complement negative staining.

However, a single antibody has a Stokes radius of ~10 nm, which reduces the effective spa­

tial resolution of this method. Recent improvements have involved the development of

genetically encoded EM labels for use in correlative light and electron microscopy (CLEM)

techniques (discussed later in this chapter).

5.2.4  TRANSMISSION ELECTRON MICROSCOPY

Biological samples can be imaged by detecting the intensity of transmitted electrons,

in transmission electron microscopy (TEM), or by the backscattered secondary electrons, in

scanning electron microscopy (SEM). TEM is valuable for probing cellular morphology in

tissues, subcellular architectures, and a range of molecular-​level samples. In TEM, the accel­

erating voltage is ~80–​200 kV, capable of generating a wide-​field electron beam at the sample

of up to several tens of microns in diameter. Contrast reagents are normally used in the form