RAPID CRYOGENIC FIXATION OF BIOLOGICAL SPECIMENS FOR ELECTRON MICROSCOPY

Abstract

This thesis describes investigations into cryofixation by the plunge-cooling technique, at ambient pressure. The objective was to characterise coolants which are commonly used for cryofixation, so that the structure and chemistry of biological specimens may be preserved in a more life-like state. The work began with the design of a suitable cooling device. This was developed further into a large test-bed apparatus which was used in both biological and methodological experiments. The large cooling apparatus demonstrated for the first time that ethane was a superior coolant under forced convection, compared to propane or Freon 22, for bare thermocouples, for exposed hydrated specimens and for metal-sandwiched hydrated specimens. Ice crystal formation was monitored in sandwiched specimens and found to correspond closely to modelling predictions. A biological application was the X-ray microanalysis of body fluids in "indicator" species of Chaetognaths, where results obtained from cryoscanning electron microscopy revealed ecophysiological differences. The use of low thermal mass supports demonstrated that good freezing can occur in the centre of specimens. A new cryomounting method was developed to load well-frozen specimens into the microscope. The effect of post-freeze processing temperature was investigated by monitoring ice crystals in red blood cells. Exposure to 213 K (-60°C) over a 48 hour period did not induce crystal growth and exposure to 233 K (-40°C) for 8 days showed minimal ice crystal damage. The progress of cryosubstitution was monitored over 48 h at 193 K ( -80°C), this showed that uranium ingressed to a depth of 320 µm which could be doubled when shrinkage was allowed for. The conclusion was that observed ice crystal damage originated during the initial freezing and not during subsequent cryoprocessing.