Home / Documents & Brochures / Technical Briefs / Cryogenic LT-SEM:

2(a). Cryogenic

Table of Contents:

1. Introduction
2. Physical Consideration
(a) Cryogenic
(b) Environmental
3. Cryo SEM Preparation System
(a) Specimen Stub & Freezing Chamber
(b) Transfer Device & Preparation Chamber
(c) Microscope Stage (SEM) & Applications

As a first approach it would seem reasonable to wish to freeze the specimen as rapidly as possible so that ice crystal damage is minimal, and to as low a temperature as practical, to avoid the possibility of sublimation. Indeed a range of specialised freezing techniques has been developed for fast freezing of very small specimens. However, for the Bulk specimens we are considering, the cooling rate will be relatively slow, and the choice of cryogen less critical.

The poor thermal conductivity of ice would also suggest that the thermal capacity of the specimen support stub might not significantly affect the cooling rate and subsequent size of ice crystals at the specimen surface. This would allow the use of a relatively large stub and the advantage of good thermal stability, while for special applications, smaller specimen mounts could still be used and catered for on the larger stub to utilise this advantage.

It would seem reasonable to consider liquid nitrogen with a boiling point of -196oC to be a potentially satisfactory cryogen; obviously this is more valid when used in its sub-cooled state (slushy nitrogen) at -210oC, to reduce the effects of an insulating gas layer being formed when freezing. It satisfies the criteria at these temperatures and pressures (between 0.1Torr and ATMS.) of inhibiting sublimation and as the process is usually one of plunging, the end point for this particular part of the process results in the specimen under liquid nitrogen, a very satisfactory storage condition.

It has been indicated that the cooling rate associated with the freezing of the specimens, with which we are concerned, may be relatively ‘slow’ due to the inherent poor thermal conductivity of the specimen, with resulting larger ice crystals and possible damage. In any case for ‘fast’ frozen specimens the containment of small ice crystals is less than some 20micrometers from the surface, a relatively small distance in the normal application of S.E.M.

As we are dealing with a range of Bulk Specimens, it is difficult to arrive at a standard Specimen Cooling Rate, which will in any case be greater at the surface. It is useful, however, to establish some indication of a base line, if for no other reason to establish a method of comparison and repeatability.

This may be reasonably obtained using a ‘bare’ thermocouple as a standard. Average Cooling rates of the order of 300oC/second are obtained, over the range 0oC-50oC with peak cooling rate of the order of up to three times this figure. While the peak cooling rate ‘applied’, should it occur at 0oC, is of primary interest with regard to the freezing of pure water, this may not normally be the case. The average ‘applied’ cooing rate obtained over the initial 0oC to -50oC could be suggested as a ‘standard’. This gives sufficient range for sensible, average measurement and calculation, with the end point satisfying the condition for freezing of solutions, experienced in Biological specimens.

By way of comparison the ‘achieved’ cooling rate for a thermocouple embedded in yeast droplet is of the order of 100oC/sec. for similar conditions for those described. The use of sub-cooled liquid nitrogen (slushy nitrogen) naturally satisfies a lower temperature than that of re-crystallisation of ice, which is considered to occur very slowly at temperatures of less than the order of -130oC. In satisfying these considerations, the structure of the specimen should be preserved satisfactorily, for both viewing and analysing.

 Page 3 of 8: 1 2 3 4 5 6 7 8