Investigating fully hydrated samples using ESEM Technology

Environmental Scanning Electron Microscopy (ESEM) technology expands the boundaries of traditional scanning electron microscopy to deliver deeper insights into all types of samples. ESEM allows imaging of samples with minimal preparation and adds variables such as hydration, thermal cycling, and introduction of gas to characterize in situ dynamic changes. Using water vapor and a temperature control stage, some of the “impossible to image samples” such as dirty (highly outgassing) and naturally hydrated samples whose properties will change with drying, are easily characterized. With full control of sample hydration, materials and life scientists are able to observe real time material interactions in solution, how humidity changes a sample, and how water interacts at the surface of samples. This moves research possibilities from pure static points in time to dynamic or in situ experimentation.

Naturally hydrated samples

With the increasing need for higher resolution, researchers move from optical techniques to electron microscopy. For some samples, this transition will necessitate removal of water and may change sample structures. Naturally hydrated specimens (>25% “free” water content) may or may not be normally covered in water, but the internal water content is what often retains a sample’s shape, and removal of the internal water can collapse and massively alter the sample surface. For some samples this can be reversible, but for others, the structural modification could lead to defects and distortion.

Pairing ESEM with a cooling stage allows maintaining any hydration state by varying temperature and pressure. The phase diagram chart below shows the relationship to humidity level.

Ideally, samples should be maintained above freezing to avoid the formation of crystalline ice which can rip apart delicate structures. In order to maintain a water hydration state, the imaging gas needs to be pure water vapor (not moist air or inert nitrogen) so the partial pressures of water will match known pure water partial pressure data to control hydration. In the ESEM, this is automated with a live display of humidity set point. Imaging naturally hydrated samples has never been easier.

Samples such as biological materials are naturally hydrated and traditionally all the water is removed by a substitution drying method. With ESEM, samples are imaged directly without substitution and drying. In Figure 3, examples of a flower petal compare adequate hydration with conditions not matching the sample’s needs — the latter resulting in structural collapse.

Of course it is possible to freeze hydrated samples and investigate with cryogenic conditions in SEM as well, however with ESEM, the general guideline is to avoid freezing. When water freezes, the molecules assume a hexagonal structure which is more open than liquid water and this space transformation will often damage and distort wet sample structures (i.e. samples with significant free water; generally >25%). For freezing without hexagonal ice, nitrogen slush plunge techniques should be used with cryogenic stages to achieve amorphous ice. ESEM allows viewing hydrated samples directly without freezing as shown in Figures 4 and 5.