SPI Critical Point Drying Apparatus
Detailed Description of the Apparatus and Service Information: Tissue
preparation
General Comments
It is suggested that the user consult the literature before deciding on any
particular technique of sample preparation. A number of different methods
are in use and the customer must decide which technique will give the best
results for his material. It must be stressed that the final result will
depend almost entirely on the preparation technique carried out before
the tissue goes into the pressure vessel.
The following technique is that recommended by Dr. A. Boyd of University
College, London. The sequence of operations outlined is very comprehensive;
in many cases it may be found possible to omit some of the stages; e.g.,
with botanical specimens it has been found that if the dehydration is
carried out very carefully, it is not necessary to pre-fix the tissue.
With difficult specimens, however, care taken in fixing and dehydration
results in a much improved result.
Note:
The whole object of the technique is to obtain a specimen which has been
dried by the critical point drying technique. During the preparatory
stages, the specimen must be given no opportunity to dry prematurely.
It must be kept wet at all times.
The complete sequence involves the following steps:
a) washing
b) fixation
c) dehydration
d) substitution with CO2 miscible liquid
e) substitution with CO2
f) heating to super-critical temperature
g) pressure release
The method involved in f) and g) has been described in the previous section.
If dehydration is done with acetone, step d) is omitted because acetone is
miscible with CO2. If acetone is being used, great care in the washing of
the acetone after use is important in order to enjoy maximum possible life
times of the bonded seals (e.g. Dowty seals).
Washing
Specimens must be washed free of mucous, blood, serum or any other
contaminant likely to be fixed on the surface. The washing medium must be
physiological, e.e. not cause any changes in shape or form of the tissue.
Fixation
If the specimen is pre-fixed in osmium tetroxide this must be isotonic.
This may then be followed by glutaraldehyde as the main fixative.
If glutaraldehyde is used as the first and only fixative, it must be left for
days rather than hours. Short periods of aldehyde fixation are not suitable
as they do not render tissues resistant to osmotic changes. Starting with
1% glutaraldehyde for 1 hour and following with 3% gives improved results.
Dehydration
Having fixed the tissue and washed it with distilled water to remove buffer
salts and other electrolytes, it must then have its water content replaced by
a liquid with a convenient critical point. This is not done directly as
water is not miscible with carbon dioxide or Freon 113. The routes in common
use are:
1) water - acetone - carbon dioxide
2) water - acetone - FreonŽ 113
3) water - ethanol - amyl acetate - carbon dioxide
4) water - ethanol - Freon 113 - carbon dioxide
5) water - ethanol - amyl acetate - Freon 113
6) water - ethanol - Freon 113
Unfortunately, the entire line of Freon chemicals has been discontinued by
their manufacturer, DuPont, because of the adverse effect this class of
chemicals was having on the environment. We would therefore recommend the
use of carbon dioxide instead of Freon (even if it was available) since the
same result could be obtained.
With regard to which method is "better", it is jut not clear from the
literature if one or the other is indeed "better". However different
researchers do seem to develop their own personal preferences for one
method over the other.
The most convenient method is the first one listed as it take the shortest
time and uses the lowest cost transition fluid. Route number 3 is also
favored as the strong smell of amyl acetate gives a good indication of
whether or not all the intermediate fluid has been removed before the
drying cycle is started.
If dehydrating to acetone, the 'Diffusion Dehydration" method can be used:
a) put tissue in 20% acetone (5ml)
b) place on shelf of desiccator with 100% acetone containing some anhydrous calcium sulfate
c) place watch glass of anhydrous CaCl2
d) pump desiccator with water pump until acetone boils
e) seal under vacuum and leave overnight
When the desiccator is opened it will be found that the tissue is in 100%
distilled acetone and has been fully and gently dehydrated.
When dehydrating to ethanol, the following method is recommended for pieces
of tissue up to 1mm in thickness:
Place in 30% ethanol for 15 minutes.
Place in 50% ethanol for 15 minutes.
Repeat with 70%, 80%, 90%, 95%, 100%, 100%, each for 15 minutes. The more
care taken with this the better the result. Rapid dehydration causes
shrinkage of tissues. Larger pieces need longer times.
Substitution
After dehydration with ethanol, further substitution to amyl acetate or
Freon 113 is necessary before putting the tissue in the pressure chamber.
Boyd recommends that the substitution should not be carried out directly,
but through graded baths (25:75, 50:50, etc.) of the two liquids. 15
minutes per step should suffice.
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Tuesday September 30, 2008
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