Ionizing collisions between electrons and the gas molecules in the specimen chamber create positive ions which are attracted onto the sample, neutralizing the specimen charge. It is therefore possible to image insulating samples without the need for metallic coating. The presence of water vapor in the chamber also means that a high relative humidity can be maintained in the microscope chamber such that samples can be imaged in a hydrated state without the need for fixation, dehydration, or critical point drying. Further details of the instrument and its operation can be found in Donald (4) and Stokes (5). In what remains of this section we highlight two aspects of ESEM imaging fundamental to the use of the protocols outlined in the following sections.

Correctly controlling the chamber conditions is critical when attempting to image hydrated specimens as the appropriate relative humidity is necessary to prevent excessive evaporation from, or condensation onto, the sample. The relative humidity at any given temperature can be calculated as the ratio of the pressure of water vapor in the chamber, to the saturated vapor pressure (SVP) at that temperature (empirically derived SVP values are tabulated in, for example (6)). Relative humidity is therefore a strong function of specimen temperature and it is vital that the temperature in the ESEM chamber is carefully controlled. This is done using a cooling stage which relies on the solid state “Peltier effect.”

The relationship between pressure, temperature, and relative humidity can be used to produce a phase diagram, delineating different thermodynamic regimes. The phase diagram for pure water is shown in Fig. 2. At conditions corresponding to points above the 100% relative humidity curve, the vapor is unsaturated and liquid water will evaporate from the specimen. If pressure and temperature conditions fall below this line, the vapor is saturated and water will condense onto the sample. Curves representing lower constant humidities may also be drawn onto such diagrams and can be used to define stable pressure and temperature conditions for specimens in the ESEM chamber. As the samples are not simply composed of pure water, the equilibrium relative humidity will certainly be less that 100%. However the exact value is sample dependent. In general it may be best to proceed cautiously by selecting the lowest temperature that the sample can tolerate, calculating the theoretical pressure corresponding to perhaps 95% relative humidity and then finding, by small adjustments, the pressure at which water begins to visibly condense. It is worth noting that for most systems the temperature set on the cooling stage may not correspond perfectly with the temperature at the surface of the specimen due to thermal transfer effects (7). Furthermore users may observe that condensation onto the sample is associated with an increase in sample temperature; this results from the liberation of latent energy due to the change of state.

Consideration must also be given as to how the desired pressure is achieved. During the pumpdown sequence, the ambient air (pressure of 760 Torr) which is in the sample chamber when the door is closed, must be removed and replaced by water vapor at the appropriate pressure. This is achieved by pumping the chamber down to a certain pressure and then flooding it with water vapor. In this way, the dry air is replaced by water vapor. However, such a pumpdown sequence exposes the sample to both evaporative and condensing conditions which, if uncontrolled, may be damaging. Cameron and Donald (8) described the pumping sequence mathematically and optimized the procedure based on two criteria. Firstly, the sample should finish the sequence at equilibrium with saturated water vapor above it; secondly, evaporation and condensation should be minimized. The protocols described in the following sections adopt these optimized pumpdown procedures unless otherwise stated.

Once the chamber has been pumped down to the desired pressure the sample can be imaged. The electron beam interacts with the specimen producing a variety of signal types which may be collected using the appropriate detector and used to generate an image. In the following sections secondary electrons (SE) (a surface-sensitive signal) are used to image plant tissue and mammalian cells, whereas transmitted primary beam electrons (a signal sensitive to compositional contrast) are used to image bacteria.

The source of the electrons used in imaging is the same as for CSEM. Secondary electrons (SE) are emitted from the sample surface due to the action of the energetic (primary) beam electrons. They provide topographic contrast (5), with peaks emitting more SE and hence appearing bright, whilst troughs appear dark. SE images of the microlandscape therefore make intuitive sense. By convention, SE are classified as electrons with energies less than 50 eV (9) and as such they can only escape from a shallow region of the surface; this means that SE images offer high resolution. The techniques outlined in Subheadings 3.1 and 3.2 use a “needle detector,” developed by Toth and Baker (10) for SE imaging at gas pressures in excess of 15 Torr. A positive bias of 800 V is applied to a needle shaped electrode. The tip of the needle should be positioned at the same height as the final pressure limiting aperture, around 10 mm away from the beam axis. The electric field strength around a sharp edge can be extremely high (11) and this configuration produces an intense ionization cascade in a region to one side of the beam axis, which extracts and amplifies the SE signal despite the presence of gas scatterers. The needle detector and gaseous amplification cascade are represented schematically in Fig. 3.

By using ESEM in combination with a transmitted electron detector, the ESEM-STEM (scanning transmission electron microscopy) technique, makes it possible to probe beneath the surface of hydrated, unprepared samples. This technique is discussed in Subheading 1.3 with reference to imaging bacterial specimens. In STEM images of unaltered bacteria, contrast arises from differences in mass-thickness, whether natural in origin (e.g., plastic accumulations in Cupriavidus necator (12), hematite crystals in magnetotactic bacteria or the gap between a bacterium’s inner and outer membrane) or artificial (dehydration-induced collapse). For bacteria which have not been metal-coated, an SE and an ESEM–STEM image look similar, with STEM typically being much sharper and having increased contrast (see Figs. 11 and 14).

In what remains of this section, we introduce three basic sample types and the specific examples that feature in the protocols described in Subheadings 2 and 3. Supporting notes can be found in Subheading 4 and the safe handling of live biological material in the microscopy lab is considered in the Appendix.