SEM preparation

After dehydration with

ethanol, samples are
Critical Point Dried
(CPD)

Purpose:
Purpose To completely dry
specimen for mounting while
maintaining morphological
details.
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
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.
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.

 Sample holders

-Keep samples separated

-Hold delicate or small

samples

-Ease of sample retrieval

 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.

 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.347

Biotechnic and Histochemistry, 1994, Williams & Wilkens vol. 69, no.4,
p192
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.
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
 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

 Sputter coating
Gold, gold palladium target
-vacuum of approx. 2 millibar
-thickness 7.5 nm to 30nm
Thermal evaporation
-Typically used for shadowing
- 2 x10-7 torr
-From coarse to fine:
Carbon, gold, chromium,
platinum, tungsten, tantalum
 Evaporation

Trough for powders/cleaning

E-beam
Used for high melting point
metals (e.g. tantalum)

Similar to create emission of

electrons from filament in
microscope

Provides highest resolution

 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

Carbon ribbon

Rotary device to
ensure uniform
coating