164 5.2  Electron Microscopy

biological components in the sample. Chemical fixation is a gradual multistage process of

sample dehydration with organic solvents such as ethanol and acetone; incubation with a

bivalent aldehyde chemical, typically glutaraldehyde or a modified variant, generates chem­

ical cross-​links that are relatively indiscriminate between different biomolecular structures

in the sample. The dehydrated, cross-​linked sample is then embedded in paraffin wax,

which is sliced with a microtome to generate sections of a just a few tens of nanometers of

thickness.

The most significant disadvantage with this multistage stage chemical preparation is

that it often generates considerable, and sometimes inconsistent, experimental artifacts.

Not least of which are volume changes in the sample during dehydration, which potentially

affect different parts of a tissue to different extents and therefore lead to sample distortion.

Cryofixation (also referred to as “snap freezing”) rapidly cools the sample using a cryogen

such as liquid nitrogen or liquid propane instead of chemical fixation, which eliminates some

of these problems. Common methods to achieve this include slam freezing, in which the

sample is mechanically positioned rapidly against a cold, flat metallic surface, and high-​

pressure freezing, which is normally achieved at a pressure of ~2000 atm.

A general method to minimize experimental artifacts is to at least aim for robustness in the

sample preparation conditions. By this, we mean that the various steps of the sample prep­

aration procedure should be optimized so that the appearance of the ultimate EM images

becomes relatively insensitive to small changes in sample preparation, for example, to select

a choice of dehydrating reagent that does not result in markedly different images to many

other reagents. In other words, this is to optimize the chemical and incubation conditions of

sample preparation to be relatively insensitive to their being perturbed.

The key aim of all sample freezing techniques is to vitrify the liquid phases of a biological

matter, principally water, to solid to minimize motion of the internal components and to

ensure that an amorphous, as opposed to a crystalline, vitreous solid results. The biggest

problem is the formation of ice crystals, which occurs if the rate of drop in temperature

is less than ~10⁴ K s⁻¹, which in practice means that freezing needs to occur within a few

milliseconds. Slam freezing can achieve this on samples, provided they are less than ~10 μm

in thickness, while high-​pressure freezing can achieve this on larger samples for up to ~200

μm thick.

Cryosubstitution can then be performed on the frozen sample, which involves low-​

temperature dehydration by substitution of the water components with organic chemical

solvents. In essence, the sample temperature is raised very slowly (over a period of a few days

typically), and as it melts, the liquid phase water becomes substituted with organic solvents;

this can facilitate stable cross-​links between large biomolecules driven by hydrophobic forces

in the absence of covalent bond cross-​links, so eliminating the need for a specific chem­

ical fixation step. Cryoembedding is then performed at temperatures less than –​10°C, and

samples can be sectioned using a cooled microtome.

Take the example of large protein complexes in the cell membrane. These include

membrane-​based molecular machines such as the flagellar motor in bacteria that rotates

to drive the swimming of bacteria and the ATP synthase molecular machine that generates

molecules of ATP (see Chapter 2). Cryofixation is an invaluable preparation approach for

these, especially when coupled to a method called “freeze-​fracture” or “freeze-​etch electron

microscopy,” which has been used to gain insight into several structural features of cells and

subcellular architectures. Here, the surface of the frozen sample is fractured using the tip of

a microtome, which can reveal a random fracture picture of the structural makeup immedi­

ately beneath the surface, yielding structural details of the cell membrane and the pattern of

integrated membrane proteins.

Aficionados of both cryofixation and chemical fixation in EM report a variety of pros and

cons for both methods, for example, on the different respective abilities of each to stabilize

the motions of certain cellular components during sample fixation. However, one should be

mindful of the fact that although EM has excellent spatial resolution and imaging contrast, all

sample preparation methods generate distortions when compared against the relatively less

invasive biophysical imaging technique of light microscopy.