SEM Preparation
Transcript of SEM Preparation
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SEM preparationSEM preparation
After dehydration withethanol, samples are
Critical Point Dried
(CPD)
PurposePurpose: 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 hadbeen 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
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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 substanceswould not help biological material, as the specimens would
suffer significant thermal damage if attempted.
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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.
MethodMethod
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Sample holdersSample 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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HexamethyldisilizaneHexamethyldisilizane
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.347
Biotechnic and Histochemistry, 1994, Williams & Wilkens vol. 69, no.4,
p192
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Mounting the specimen onto stubsMounting 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 electronsfrom crevices
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Coating the Sample
-Using OsO4 as fixative (biological)
-Painting a grounding line with silver or carbon paste
-Coating with nonreactive metal or carbon
a) Increased conductivity
b) Reduction of thermal damage
c) Increased secondary and backscattered electron emission
d) Increased mechanical stability
Accomplished by:Accomplished by:
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Gold, gold palladium target
-vacuum of approx. 2 millibar
-thickness 7.5 nm to 30nm
Sputter coatingSputter coating
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Thermal evaporationThermal 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-beamUsed 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
Good vacuum required
Carbon rod may need outgassing
Do not look directly at heated electrodes
For samples in SEM where
x-ray information is needed.
TEM grids needing extra
support
Support for replicas
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Rotary device to
ensure uniform
coating
Carbon ribbon