Imaging of Hydrated Samples

Imaging fully hydrated samples is a well-established and mature technique (2,3,15). It requires two components, an ESEM variant of VPSEM (to operate at the higher chamber gas pressures), and a specimen cooling stage, usually Peltier-cooling based (see Fig. 6). A recent detector development (13) that can operate at high chamber gas pressures has made the latter unnecessary but only with one of the commercial ESEM variants and also only at higher primary beam energies (20 kV).

The imaging procedure is simple in plan; operate under conditions at or close to 100% relative humidity. These conditions are achieved by using water vapor as the imaging gas and by operating at pressure and temperature conditions that lie anywhere on the water saturation curve (see Fig. 7). The usual practice is to operate with the sample close to, but always above, the freezing point. This allows the minimum gas pressure to be used and thus to minimize

Fig. 6. An in situ photograph and operational schematic, insert (A), of a simple homemade Peltier cooling stage used for imaging of fully-hydrated samples in an ESEM. The stage has a 25 x 25-mm surface area and allows large samples to be examined. The example image, insert (B), shows fully hydrated diatoms with intact processes in situ on the surface of an algal strand of a modern stromatolite from Hamelin Bay, Western Australia.

Fig. 6. An in situ photograph and operational schematic, insert (A), of a simple homemade Peltier cooling stage used for imaging of fully-hydrated samples in an ESEM. The stage has a 25 x 25-mm surface area and allows large samples to be examined. The example image, insert (B), shows fully hydrated diatoms with intact processes in situ on the surface of an algal strand of a modern stromatolite from Hamelin Bay, Western Australia.

the primary beam scatter. The limiting pressure is the equilibrium vapor pressure over ice at 0°C is 600 Pa (4.5 torr). Typically water vapor pressures around 700 to 800 Pa (~5-6 torr) and specimen temperatures around 3°C are used.

2.3.1. Practical Procedure

1. Precool the sample on the stage to usually approx 10 to 12°C (remember that cooling is from the base and thick samples will have some degree of thermal inertia).

2. Place several droplets of water around the sample (these are sacrificial and if conditions are above equilibrium then evaporation from these droplets will cool the sample down to equilibrium).

3. Evacuate the sample chamber ("flush" or inject the sample chamber with water vapor as soon as allowed by the vacuum system—this will exchange the air in the sample chamber for water vapor before the sample dehydrates. Remember the equilibrium conditions relate to the water vapour pressure only).

Temperature (DC)

Fig. 7. The water saturation curve and relative humidity P-T regimes (source data from http://hyperphysics.phy-astr.gsu.edu/hbase/kinetic/watvap.html#c1). The sample hydration management from laboratory ambient through to ESEM imaging conditions is superimposed on the water saturation curve.

4. Use the gas pressure control to fine tune the conditions (pressure changes are rapid whereas the temperature control is governed by the cooling rate of the stage and sample thickness and thermal characteristics).

The ideal path of changing conditions from laboratory ambient to stabilized 'wet' imaging conditions is shown in Fig. 7.

2.3.2. Optimizing Image Quality Use

1. The smallest final aperture size (this restricts the field of view but it protects the column vacuum and minimizes beam scatter in the second pressure region behind this aperture).

2. The shortest possible working distance. (A simple rule of thumb is that if a gap is visible between your sample and the GSED, via a ccd-based chamberscope or viewing port, then you are too far away!)

3. A primary beam current that gives an acceptable image (usually this will be significantly greater than "normal" for your microscope because a high proportion of the primary beam is scattered under these conditions and, therefore, to achieve conventional SNR levels, these higher primary beam currents are required. The beam resolution will degrade in conventional SEM columns but this should be offset by the use of the short working distance).

4,000

3,500

3,500

4,000

4. An accelerating voltage approx 5 to 10 kV. (The lower end is preferable to maximize surface detail in the GSE images. At 5 kV, the primary beam scattering is very severe and operating at the shortest possible working distance and high beam current are essential.)

All ESEM with cooling stages will provide high-quality images of "wet" samples once a minimal level of operator skill is achieved. With care, samples can be introduced under a thin film of water than can then be evaporated away to expose the surface. This technique is useful for extremely sensitive samples but risks freezing the sample because of the cooling effect of the evaporation. The rule here is to proceed very slowly, that is, reduce the pressure by only 10 to 20 Pa from equilibrium and then be patient. Never adjust the temperature via the cooling stage unless you are certain that it is incorrectly set as the response is slow and surface damage (dehydration) can occur very quickly.

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