The restriction on the thickness of a sample for high
resolution TEM at normal voltages is severe - typically 100nm. The specimen
must transmit sufficient electrons to form an image, with minimum energy loss
and it must be stable under electron bombardment in a high vacuum.
Most materials
are initially unsuitable for TEM and so must undergo one of
a variety of procedures resulting in a specimen that can be
mounted on a grid (a 3mm diameter circular metallic mesh, made
by a refined electroplating technique, usually of copper).
The grid acts as a basic support and also to conduct away the
current and heat to the body of the specimen holder. For most
specimens, the grids are coated with a thin plastic film and
a layer of evaporated carbon a few nanometres thick which acts
as extra support for delicate, particulate matter or sections.
 For biological samples, we most regularly need to cut
thin sections if we want to look at structures inside cells.
This involves first "fixing" the tissue, typically using aldehydes and/or osmium, followed
by dehydration through a series of alcohols before infiltrating the material
with a resin which we can later polymerise into a hardened "block" to provide
mechanical support during sectioning and subsequent handling. This process is
known as "embedding". Two types of embedding media
are in widespread use in EM: the acrylics and the epoxy resins.
Epoxy resins are known to have good sectioning properties,
harden uniformly, change very little in volume during curing
and are very stable in the electron beam. Therefore they are
often the resin of choice for the best ultrastructural observation.
However, acrylic resins have a very low viscosity, even at
the lower temperatures required for immmunocytochemistry (see low temperature embedding),
and are miscible with water to varying degrees depending on the type used.
They also have lower toxicity than the epoxy resins and although the
preservation of ultrastructure is not as good, especially of lipids, they have
become the resin of choice for EM (and for some LM applications) at JIC. We
now typically use LR White (from the London Resin company) for all our
embedding protocols.
 Once an embedded "block" of material has been prepared,
the size of the block "face" must be reduced by trimming, first by hand and
then on an ultramicrotome (instrument for cutting ultra-thin sections for the
TEM), so that the resultant sections will fit within the diameter of a grid.
The trimmed block face is then mechanically advanced towards the cutting edge
and falls past a stationary knife (made of glass or diamond) to shear a thin
slice away from the front. The cut sections float on the surface of a "boat"
of water attached behind the cutting edge and are picked up
from the surface with a grid held in fine forceps.
The sections can be contrast stained with uranyl acetate
and lead citrate if only ultrastructural examination is required,
or may be ummunolabelled to locate distribution of certain
epitopes using gold-conjugated antibodies.
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