ESEM (Environmental Scanning Electron Microscopy) enables the investigation of native, hydrated and uncoated plant surfaces without further sample preparation and the in situ observation of dynamic processes at SEM resolution. A selection of representative plant samples and applications will be presented to show that ESEM is a versatile tool in plant science.

It is difficult to imagine plant sciences without the comprehensive knowledge on plant surfaces achieved by SEM work. For conventional SEM samples have to be vacuum stable and conductive and for a lot of sample types these demands mean laborious preparation (chemical fixation, dehydration, critical point drying and metal coating) prior to SEM analysis. This processing, however, might change the pristine surfaces.

ESEM

Environmental SEM (ESEM) enables the investigation of nonconductive (uncoated) and hydrated samples without preceding preparation steps. This is achieved by the presence of a gas in the chamber that takes part in charge prevention and secondary electron detection (specific SE detectors) via gas cascade amplification [1]. In most cases this gas is water vapor with the positive effect for biological samples that relative humidity in the chamber can be adjusted by choosing appropriate parameters of chamber pressure and sample temperature [2, 3].

The ESEM can be operated in two modes [4]. In the wet mode (wet-ESEM) it is possible to control the thermodynamic stability of moist and liquid specimens by an appropriate chamber pressure in combination with sample cooling. A vapor pressure of 5.2 torr (690 Pa) at 5°C corresponds to 80% RH. These chamber conditions effectively prevent sample dehydration. However, a long path of the primary beam through the water vapor would result in a dramatic deterioration of the signal [2, 3]. To minimize this pressure limiting apertures separate the chamber from the high vacuum in the column with the consequence of a reduced field of view at low magnification (fig. 2). In the low vacuum (LV-ESEM) the chamber pressure is limited to 1 torr (133 Pa). This gas pressure is sufficient to mitigate negative charging but not sufficient enough to prevent dehydration of moist or wet samples since 1 torr corresponds to 5% RH at room temperature.

However, no pressure limiting apertures are necessary, the interactions with the primary beam do not deteriorate the signal, and these chamber conditions allow the simultaneous use of a standard backscattered electron detector (BSED) [4].

Two ESEM Modes Offer a Broad Range of Applications in Plant Science

Trichomes are representative examples for ESEM investigations of more or less dehydration resistant plant samples (fig. 2). These structures originate from epidermal cells and develop outwards on the surface of various plant organs. The subset of glandular trichomes produces secondary plant substances (e.g., essential oils). Epidermal cells and the detailed surface structures of the trichomes can be extensively investigated in both ESEM modes but there are significant differences concerning secretion processes and products. Secretions products are visible as smooth and thin layers on the cell surfaces using wet-ESEM. Exposition in LV-ESEM results in dehydration and a viscous appearance of these surface layers. During preparation for conventional SEM they vanish and only by chance some residues remain (fig. 2).

Secretion products also play an important part on the wet stigmata of certain plant groups providing optimal germination conditions for pollen (fig. 3). This secretion layer consisting of primary lipids and proteins can be investigated in wet-ESEM as well as in LV-ESEM. In the latter, however, investigation time is significantly restricted because of progressive dehydration. It is possible to simultaneously use a BSED to identify inorganic depositions (calcium crystals). The actual stigma surface with lots of papillae is only visible after fixation and dehydration for conventional SEM. Other plant groups are characterized by dry stigmata often consisting of papillae with only a thin cuticle layer (fig. 1). These surfaces, however, are extremely sensitive to dehydration as well as to beam damage, even if the thermodynamic stability is efficiently controlled [4]. Sorting out these problems ESEM will provide an opportunity to study the highly dynamic process of pollen germination in vivo at SEM resolution.

Dynamic Investigations

The possibility to investigate dynamic processes is a unique feature of ESEM [2, 3, 4]. Dynamic investigations are not possible with conventional SEM where samples have to be fixed and laboriously processed. The process of anther opening was intensively studied using LV-ESEM [4, 5, 6]. Another application is the mounting of a tensile stage in the SEM chamber that allows the direct observation of structural changes during tensile testing and enables studying mechanical properties of plant samples in situ [4, 7].