There are "alternative" modes of imaging using the SEM that allow you to look at difficult to process samples. The conventional method of processing relies on chemical fixation and stabilization of the sample. The "alternative" methods are attempts to look at specimens in a more "natural" state. The "alternative" techniques that are referred to are cryo-SEM, and "nature" (a.k.a. variable pressure) SEM.

Cryo-SEM

Cryo-SEM allows one to view speciments that do not lend themselves to chemical fixation. Examples of these types of specimens would be: endothelium with mucoid layers intact, novel fractured surfaces, elemental analysis of mobile electrolytes, ice cream, etc. The SEM can be interfaced with a cryo attachment that performs freezing, fracturing, and coating of the specimen if desired. The specimen is held at liquid nitrogen temperatures while viewed. This prevents outgassing which could degrade the vacuum.

Cryo-SEM, while allowing one to look at difficult specimens, also has some limitations. Due to freezing artifacts (ice crystal growth), resolution and useful magnification is limited. As a general rule, 20,000 times magnified would be the limit.

Nature SEM

The latest technological advance are the "environmental" or "nature SEM's. The instruments allow one to vary the vacuum level in the specimen chamber. By having a poorer vacuum around the specimen, it will not outgass as rapidly. Even with a compromised vacuum, outgassing will occur. Therefore, robust specimens are more easily viewed. A few examples of robust samples might include: plant leaves, cartilage and bone. Theoretically, these specimens can be placed directly into the instrument without any preparation.

As with cryo-SEM, resolution is limited in environmental SEM. The signal being used to form the image is back-scattered electrons. By definition. BSEs give poor spatial (topographically) information. Magnifications in excess of 20,000 will produce images of poor quality.

In conclusion, cryo-SEM and NSEM can be used to gather information from hard to prepare specimens. Sometimes, it is the only approach to imaging some specimens.