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3. Practice

Table of Contents:

1. Introduction
2. Principle
(a) Sublimation Considerations
(b) Freezing Considerations
3. Practice

If we consider that we can freeze dry at -60oC, having satisfactorily fast frozen the specimen, then referring to Fig. 3 we can establish the conditions for freeze-drying.

Freeze-drying Conditions

Figure 3.

These conditions (-60oC at Saturated Vapour pressure of ice (Ps),) can be readily accommodated using Thermoelectric Cooling (Peltier), and rotary vacuum pump pressures (Pv) to ensure the partial pressure of water (Pp) is not limiting in the freeze-drying process. This is enhanced by the use of a Water Vapour Trap, which may be in the form of a Molecular Sieve that is a 'spectator' not involving any chemical reaction or the more efficient desiccant, Phosphorous Pentoxide (P205).

Emitech Freeze Drier Chamber (cross section)

Figure 4: Cross sectional view of an Emitech Freeze Drier Chamber illustrating the Peltier Stage which can be used in normal manner or with the Capsule Transfer System

In practice, after the initial sublimation of the outer layers, diffusion through the tissue is probably the more determining factor in the drying process, with much less dependence on the removal of water to the Vapour Trap.

It could be considered that after the initial drying at -60o, increasing the temperature to -40oC, may enhance the latter stages of drying. A possible sequential schedule could be 4 hours at -60o, 4 hours at -40oC. In a design brief it may be apparent that we want to satisfy several conditions and maintain maximum system flexibility.

Emitech K750X Freeze Drier

Figure 5: Emitech K750X Freeze Drier Peltier System. Indicating Dual Time and Temp Modules and Ramp Control warm up facilities.

  1. Prepare and Freeze the specimen in a suitable Cryogen.
  2. Satisfactorily transfer the specimen to the drying unit.
  3. Select two differing Drying Temperatures, for two different time periods, either independently or sequentially.
  4. Vacuum Resin Embed.
  5. Sputter Coat.
  6. Carbon Coat.

(These facilities should be readily available without compromise to the system performance and user ergonomics.)

Scanning Micrograph of an Aphid

Figure 6: A Scanning Micrograph of an Aphid, Freeze Dried using the K750 system and Sputter Coated with Gold. Aphids secret wax, ribbons of which show good preservation, normally 'LOST' in Drying methods involving solvents.

In practice, temperatures of -60oC, as found in the K750X, have been found to give reasonable results. For particular applications, however, it can be necessary to freeze dry at temperatures below -80oC with lower sublimation rates or delicate specimens. This requires a better vacuum than can be obtained using a rotary vacuum pump.

Emitech K775X Turbo Freeze

Figure 7: K775X Turbo Freeze.

The K775X Turbo Freeze Drier operates at temperatures below -80oC, controllable in the range -120oC to +40oC and has a programmable multi-segment sequence controller with 10 times and 10 temperatures. It employs a Turbo-Molecular pumping unit, backed by a Rotary Vacuum pump.

Freeze-drying Conditions

Conditions used for freeze drying sections of ASM cells in a temperature controlled turbomolecular freeze drier (vacuum better than 10-2 mbar).


ASM Preparations

Figure 8: a) Preparation of ASM which has been Cryo-Sectioned and freeze dried at low vacuum, note re-hydrated appearance. b) Preparation of ASM which has been freeze dried under controlled conditions (using Emitech K775X), sub-cellular structures such as nucleus (n), mitochondria (m) and RER can be seen, Marker=1.0m. Photos courtesy of Dr.Alice Warley, Division of Opthalmology, The Rayne Institute,U.M.D.S., Lambeth Palace Road, London, SE1 7EH.

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