Goals of Specimen Preparation

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Goals of Specimen Preparation Observe specimen
near natural state as possible. Preserv
ation of as many features as possible. Avoid
artifacts (changes, loss or additional
information) Render specimen stable for
examination in environment of SEM.
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Tissue
Standard Preparation
TEM
SEM
Chem. Fixation
Cryo Fixation
Chem. Fixation
Cryo Fixation
Rinse/store
Substitution
Rinse/store
En bloc staining
Cryo- sectioning
Dehydration
Dehydration
Dehydration
Drying
Resin infiltration
Mounting
Sectioning
Coating
Post staining
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SEM preparation
After dehydration with ethanol, samples are
Critical Point Dried (CPD)
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Critical point drying (CPD)
Purpose To completely dry specimen for mounting
while maintaining morphological details.
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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 cold
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
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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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Used for high melting point metals (e.g.
tantalum) Similar to create emission of
electrons from filament in microscope Provides
highest resolution
E-beam
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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
Carbon Rods