SEM preparation

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SEM preparation
After dehydration with ethanol, samples are
Critical Point Dried (CPD)
Purpose To completely dry specimen for mounting
while maintaining morphological details.
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If the temperature of liquefied gas is increased
the meniscus becomes flatter indicating a
reduction in the surface tension. If the surface
tension becomes very small the liquid surface
becomes very unsteady and ultimately disappears.
When this 'critical point' is reached, it is
possible to pass from liquid to gas without any
abrupt change in state. If a specimen had been in
the liquid it would have experienced a transition
to a 'dry' gas environment without being in
contact with a surface, avoiding the possibility
of the damaging effects of surface tension.
This is termed Critical Point Drying (C.P.D.) the
basis of which are the classic experiments
carried out over 100 years ago during
investigations on the liquefaction of gasses.
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Initial investigations were CO2 as will be
apparent from Figure 2 - table of Critical
Constants for some common substances. The
critical conditions of other substances would
not help biological material, as the specimens
would suffer significant thermal damage if
attempted.
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Method
1) Water exchanged for ethanol. 2) Ethanol
exchanged for liquid CO2 (transitional
fluid). 3) CO2 brought to critical point (31.1 C
and 1,073 psi), becomes dense vapor phase. 4)
Gaseous CO2 vented slowly to avoid
condensation. 5) Dry sample ready for mounting.
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Sample holders
-Keep samples separated -Hold delicate or
small samples -Ease of sample retrieval
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Freeze Drying
-Sample is quick frozen in liquid nitrogen
(LN2). -Placed in vacuum evaporator on frozen
block (approx. -190 C). -Left under vacuum for
several days to sublimate water. -Mounted and
coated.
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Hexamethyldisilizane
HMDS is a chemical method of drying the
sample Primarily used with insects, larger
fleshy tissues, soft invertebrates, etc... HMDS
is a strong irritant and volitile
(flammable). Brief protocol -After fixation -
ethanol dehydration to 100 -Transition from
ethanol to HMDS -Two changes of pure HMDS -Left
overnight in dessicator with silica gel
Stain Technology, 1983, Williams Wilkins vol.
5, NO. 6, p.347Biotechnic and Histochemistry,
1994, Williams Wilkens vol. 69, no.4, p192
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Mounting the specimen onto stubs
Stubs are specimen holders specific for the
instrument being used (e.g. Zeiss or Hitachi SEM)
Specimen is held to stub by conductive tape,
paste or glue.
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Conductivity of Samples Charging results
in deflection of the beam deflection of some
secondary electrons periodic bursts of secondary
electrons increased emission of secondary
electrons from crevices
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Coating the Sample
a) Increased conductivity b) Reduction of
thermal damage c) Increased secondary and
backscattered electron emission d) Increased
mechanical stability
Accomplished by
-Using OsO4 as fixative (biological) -Painting a
grounding line with silver or carbon
paste -Coating with nonreactive metal or carbon
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Sputter coating
Gold, gold palladium target -vacuum of approx. 2
millibar -thickness 7.5 nm to 30nm
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Thermal evaporation
-Typically used for shadowing - 2 x10-7
torr -From coarse to fine Carbon, gold,
chromium, platinum, tungsten, tantalum
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Evaporation
Trough for powders/cleaning
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E-beam
Used for high melting point metals (e.g.
tantalum) Similar to create emission of
electrons from filament in microscope Provides
highest resolution
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Carbon Coating
For samples in SEM where x-ray information is
needed. TEM grids needing extra support Support
for replicas
Good vacuum required Carbon rod may need
outgassing Do not look directly at heated
electrodes
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Carbon ribbon
Rotary device to ensure uniform coating