Cryo-SEM
Unlike many other cryo-techniques, for observation of
plant material, low temperature scanning electron microscopy has established
itself as a major tool in it's own right. Cryo-SEM was first performed in the
1960's but it wasn't until the 1980's that commercially available systems made
it a routine facility in many laboratories.
Cryo-SEM involves the examination of
biological material at below ambient temperature (typically between -100oC
and -175oC), allowing the life-like appearance of the sample to
be preserved and recorded in the fully hydrated and chemically unmodified state.
First, the sample is cryo-fixed, generally by plunging it into sub-cooled
nitrogen (nitrogen slush) close to the freezing point of nitrogen at -210oC,
then the sample is transferred in vacuo to the cold-stage of the SEM
cryo-preparation chamber, where fracturing can be performed if necessary. After
sputter coating with metal (usually gold or platinum), the sample is transferred
into the SEM chamber, where it remains frozen during imaging on another cold-stage,
cooled by nitrogen.
This method is quick, relatively easy and cheap, and provides
superior images to those of critical point dried samples in the SEM. It is
also the only method that can be used successfully if one requires visualization
of delicate or labile structures, such as spores, that would be lost during
specimen processing in liquids. It also allows the study of epicuticular waxes,
which would be solubilized during the standard dehydration series used in convential,
ambient temperature SEM.
Although the freezing rate for these specimens is
not particularly fast, SEM is commonly used for the study of surface structures,
so that "slow" freezing is actually not a problem. In
fact, for some samples, such as root hairs, this can be of benefit. It is for
reasons such as these, that cryo-SEM has become the method of choice for a wide
range of botanical specimens. Another advantage, particularly over the replica
technique, is that large areas of an intact specimen can be studied, both at
low and high magnification.
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