BRIGHT OTF6000, Руководство пользователя

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Bright Instruments 38
3.1 BASIC FREEZING TECHNIQUES
As a general rule, the quicker tissues are
frozen the better the results will be. The
aim is to minimise the damage caused by
ice crystal artefact, and the more rapid the
transition from liquid phase to solid phase,
the smaller the ice crystals will be. The
susceptibility to damage varies between
tissue types, with certain tissues (e.g. muscle)
being very easily damaged. Furthermore, the
nature of the work may dictate the level of
tissue disruption which is acceptable. In all
cases it is important to keep the specimen
size minimal, i.e. at least one dimension should
be no more than a few millimetres.
3.1.1 LIQUID NITROGEN
With a boiling point of -196ºC, LN2 is an ideal
freezing agent. Tissues may be immersed
directly, or more frequently a solvent, such as
hexane, is pre-cooled in LN2 then specimens
are immersed in the solvent.
3.1.2 CARBON DIOXIDE - GAS
The traditional method of freezing. A blast
of gas from a CO
2
cylinder will achieve
temperatures around -60ºC. Care must be
taken with pressurised gas, and the risk of
creating an aerosol of potentially infectious
tissue particles must be borne in mind.
3.1.3 CARBON DIOXIDE - SOLID
This can be used in a bath of solvent, such
as hexane, to achieve temperatures around
-60ºC. Safer than CO
2
gas, solid CO
2
(cardice)
remains one of the most popular methods of
freezing.
3.1.4 FREEZER PLATES
Some cryostats incorporate special cold plates
for rapid freezing, covering the temperature
range -45ºC to -60ºC. The OTF cryostat has
a standard quick freezer plate that operates
at approximately 10ºC below chamber
temperature.
3.2 FITTING THE SPECIMEN
For setting up tissue, proceed as follows:
a. Remove the frost elimination cover from the
quick freezer plate.
b. Make sure that the object holder is above
0ºC, if it is at room temperature that is fine
and it just takes a little longer. It is important
to note that embedding compound does not
stick to a surface that is below 0ºC.
c. Place a small amount of embedding
compound on the top surface of the object
holder and make sure that it covers all of it in
order to get the maximum area for adhesion.
Try to avoid spilling embedding compound
over the side.
d. Place the object holder in one of the semi-
circular recesses of the quick freezer plate so
that the machined slot fits with the top of the
object holder a few mm above the level of the
plate. Alternatively, if the quick freezer plate
has frosted over too much then object holders
may be stood on the top of it.
3. Cutting & Collecting
Sections