Section 19.6.4.3. Specimen preparation

The goal in preparing specimens for cryomicroscopy is to keep the biological sample as close as possible to its native state in order to preserve the structure to atomic or near atomic resolution in the microscope and during microscopy. The methods by which numerous types of macromolecules and macromolecular complexes have been prepared for cryo EM studies are now well established (e.g. Dubochet et al., 1988). Most such methods involve cooling samples at a rate fast enough to permit vitrification (to a solid glass-like state) rather than crystallization of the bulk water. Noncrystalline biological macromolecules are typically vitrified by applying a small (often <10 µl) aliquot of a purified ~0.2–5 mg ml⁻¹ suspension of sample to an EM grid coated with a carbon or holey carbon support film. The grid, secured with a pair of forceps and suspended over a container of ethane or propane cryogen slush (maintained near its freezing point by a reservoir of liquid nitrogen), is blotted nearly dry with a piece of filter paper. The grid is then plunged into the cryogen, and the sample, if thin enough (∼0.2 µm or less), is vitrified in millisecond or shorter time periods (Mayer & Astl, 1992; Berriman & Unwin, 1994; White et al., 1998).

The ability to freeze samples with a timescale of milliseconds affords cryo EM one of its unique and, as yet, perhaps most under-utilized advantages: capturing and visualizing dynamic structural events that occur over time periods of a few milliseconds or longer. Several devices that allow samples to be perturbed in a variety of ways as they are plunged into a cryogen have been described (e.g. Subramaniam et al., 1993; Berriman & Unwin, 1994; Siegel et al., 1994; Trachtenberg, 1998; White et al., 1998). Examples of the use of such devices include spraying acetylcholine onto its receptor to cause the receptor channel to open (Unwin, 1995), lowering the pH of an enveloped virus sample to initiate early events of viral fusion (Fuller et al., 1995), inducing a temperature jump with a flash-tube system to study phase transitions in liposomes (Siegel & Epand, 1997), or mixing myosin S1 fragments with F-actin to examine the geometry of the crossbridge powerstroke in muscle (Walker et al., 1999).

Crystalline (2D) samples can fortunately often be prepared for cryo EM by means of simpler procedures, and vitrification of the bulk water is not always essential to achieve success (Cyrklaff & Kühlbrandt, 1994). Such specimens may be applied to the carbon film on an EM grid by normal adhesion methods, washed with 1–2% solutions of solutes like glucose, trehalose, or tannic acid, wicked with filter paper to remove excess solution, air dried, loaded into a cold holder (see below), inserted into the microscope, and, finally, cooled to liquid-nitrogen temperature.

Specimen preparation for cryomicroscopy is, of course, easier to describe than perform (`the Devil is in the details'). Success or failure depends critically on many factors such as: sample properties (pI, presence of lipids etc.); sample concentration (usually much higher than that needed for negative staining) and temperature; stability, age and wetting properties of the support film and need for glow-discharging (Dubochet, Groom & Müller-Neuteboom, 1982) or use of lipids (Vénien-Bryan & Fuller, 1994) to render the film hydrophilic or hydrophobic; time of sample adsorption to the film; humidity near the sample; extent of blotting and time elapsed before freeze-plunging; and concentrations and types of solutes present in the aqueous sample or the need to remove them (Trinick & Cooper, 1990; Vénien-Bryan & Fuller, 1994). Lastly, the experience and persistence of the microscopist may be critical in judging which factors are most important. Fortunately, cryo EM has evolved long enough to demonstrate that a wide variety of fragile macromolecular assemblies can be preserved and imaged in a near-native state.

Alternative procedures exist for each step of sample preparation. Particulate specimens (i.e. single particles) are usually prepared on holey carbon films, which are sometimes glow-discharged to enhance the spreading of the specimen. Continuous carbon films, carbon-coated plastic films and even bare grids (Adrian et al., 1984) have been used as supports for different specimens. Several techniques and freezing devices have been developed for producing uniformly thin, vitrified samples (e.g. Taylor & Glaeser, 1976; Dubochet, Chang et al., 1982; Bellare et al., 1988; Dubochet et al., 1988; Trinick & Cooper, 1990). All subsequent steps, up to and including the recording of images in the microscope (Section 19.6.4.4), are carried out in a manner that maintains the sample below −170 °C to avoid devitrification, which occurs at ∼−140 °C and leads to recrystallization of the bulk water to form cubic ice (Dubochet, Lepault et al., 1982; Lepault et al., 1983).

These steps include transfer of the grid from the cryogen into liquid nitrogen, where it may be stored indefinitely, and then into a cryo specimen holder that is cooled with liquid nitrogen (e.g. Dubochet et al., 1988). The cold holder is rapidly but carefully inserted into the electron microscope to minimize condensation of water vapour onto the cold holder tip, otherwise such water ruins the high vacuum of the microscope and also contaminates the specimen. Indeed, because the cold specimen itself is an efficient trap for any contaminant, most cryo EM is performed on microscopes equipped with blade-type anticontaminators (e.g. Homo et al., 1984) that permit individual EM grids to be viewed for periods of up to several hours. Also, cryo holders are subject to greater instabilities than conventional, room-temperature holders owing to the temperature gradient between microscope and specimen and because boiling of the liquid-nitrogen coolant in the Dewar of the cold holder transmits vibrations to the specimen. The maximum instrumental resolving power of most modern microscopes (∼0.7–2 Å) cannot yet be realized with commercially available cold holders, which promise stability in the 2–4 Å range.