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Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possible. Avoid artifacts (changes, loss or additional information) Render specimen stable for examination in environment of SEM. Standard Preparation. Tissue. TEM. SEM.
Observe specimen near natural state as possible.
Preservation of as many features as possible.
Avoid artifacts (changes, loss or additional information)
Render specimen stable for examination in environment of SEM.
After dehydration with ethanol, samples are Critical Point Dried (CPD)
Purpose: To completely dry specimen for mounting while maintaining morphological details.
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.
-Keep samples separated
-Hold delicate or small
-Ease of sample retrieval
-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.
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).
-After fixation - ethanol dehydration to 100%
-Transition from ethanol to HMDS
-Two changes of pure HMDS
-Left overnight in dessicator with silica gel
“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.
Charging results in:
deflection of the beam
deflection of some secondary electrons
periodic bursts of secondary electrons
increased emission of secondary electrons from crevices
a) Increased conductivity
b) Reduction of thermal damage
c) Increased secondary and backscattered electron emission
d) Increased mechanical stability
-Using OsO4 as fixative (biological)
-Painting a “grounding line” with silver or carbon paste
-Coating with nonreactive metal or carbon
Gold, gold palladium target
-vacuum of approx. 2 millibar
-thickness 7.5 nm to 30nm
-Typically used for shadowing
- 2 x10-7 torr
-From coarse to fine:
Carbon, gold, chromium, platinum, tungsten, tantalum
Trough for powders/cleaning
Similar to create emission of electrons from filament in microscope
Provides highest resolution
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
Rotary device to
ensure uniform coating