SEM preparation

1. SEM preparation After dehydration with ethanol, samples are Critical Point Dried (CPD) Purpose: To completely dry specimen for mounting while maintaining morphological details.

2. 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.

3. 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.

4. 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.

6. Sample holders -Keep samples separated -Hold delicate or small samples -Ease of sample retrieval

7. 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.

8. 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

9. 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.

10. 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

12. 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

13. Sputter coating Gold, gold palladium target -vacuum of approx. 2 millibar -thickness 7.5 nm to 30nm

14. Thermal evaporation -Typically used for shadowing - 2 x10-7 torr -From coarse to fine: Carbon, gold, chromium, platinum, tungsten, tantalum

15. Evaporation Trough for powders/cleaning

16. E-beam Used for high melting point metals (e.g. tantalum) Similar to create emission of electrons from filament in microscope Provides highest resolution

17. 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

18. Carbon ribbon Rotary device to ensure uniform coating