Environmental (ESEM) and low vacuum scanning electron microscopy (LVSEM)
About this technique
Scanning electron microscopes can be operated in a variety of vacuum states (or modes): high vacuum for conventional SEM and low vacuum (reduced vacuum or variable pressure) and environmental SEM (ESEM) for viewing samples in their natural state without the need for desiccation.
For viewing samples using a high vacuum mode, samples must be completely dry and conductive. Nonconductive samples are usually coated with a thin layer of gold, platinum or carbon. However this can limit further use of the sample. In the low vacuum mode and in ESEM, samples can be viewed without being conductive. They do not need to be coated or to have extensive sample preparation such as fixation and dehydration. The absence of coating is ideal when compositional analysis using spectroscopic techniques is required as it can be undertaken on the native sample without being compromised by the coating material.
An advantage of using the low vacuum (LV) mode in SEM is that the pressure can be adjusted in the sample chamber until the artefact of “electron charging” is removed from images. This charging artefact occurs when electrons from the electron beam build up in a nonconductive sample. The extra electrons then discharge from the sample unpredictably, causing lines and streaks on the generating image, or they repel the beam, causing jumps in the image or the appearance of black patches.
In LV mode samples are imaged using backscattered electron imaging (BSE) and therefore, nonconductive, uncoated samples viewed in this way can provide information about composition via the contrast of the image: whiter regions have a higher average atomic number than darker regions.
The LV mode can also be used to freeze-dry samples. The sample is placed on a mount, plunged into liquid nitrogen and then placed on the SEM machine stage. The chamber is then pumped free from air (evacuated). It takes about 10 minutes to remove the water from the frozen sample, as it warms up. It is then ready for viewing. This technique works best on samples that have some basic structural integrity, such as plant tissue.
A wide variety of samples can be viewed and analysed using the LV mode; for example insect tissues, plant material, biological and non-biological polymers such as hydrogels, particulate samples, and geological materials.
It should be noted that while LV mode allows adjustment of the pressure within the sample chamberthis is not to the degree achieved in an ESEM.ESEM, like LV SEM, has the advantage over conventional SEM of being able to image samples in their natural state. It can be used in what is termed the wet mode where samples containing water can be viewed without the need for desiccation. Relative humidity (RH) can be controlled within the chamber by adjusting the temperature of the conventional stage (±20º C) along with the pressure. For example a relative humidity of 100% can be achieved by combination of low temperature (e.g. 4º C) and high water vapour pressure (e.g. 6.1 Torr). The advantage of using 100% RH is that the sample is not being dehydrated as it is being imaged. Water can also be condensed on the samples by going above 100% RH.
Dynamic experiments can also be carried out on wet samples in real time, involving heating on a specialised hot-stage, anywhere up to 1500º C, cooling, wetting and drying. The samples can be imaged while these dynamic processes are occurring. Some examples of experiments that can be undertaken in the ESEM include the determination and imaging of melting dynamics for physical science materials; determination of crystallisation dynamics; and imaging of biological processes, for example pollen tube growth in real time through wetting of pollen.