Burke, Patricia Virginia (1971) Freezing phycomyces sporangiophores in superfluid helium for ultrastructure studies. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-08082006-142417
Knowledge of the ultrastructure of the growing zone of Phycomyces sporangiophores is a desirable adjunct to studies of their sensory physiology. This includes discovery of the type of organelles present and their spatial arrangement. Chemical fixation preserves some of the cell's organelles satisfactorily, but considerable disruption and dislocation occur. Physical fixation by freezing should preserve the spatial distribution of organelles within the cell if the freezing is rapid enough to prevent ice crystal formation. Ice crystal inhibitors do not penetrate the cell satisfactorily, so that crystal size is determined only by the freezing rate.
Liquid He II exhibits a quantum mechanical mechanism of heat transfer which is much more efficient than the normal, classical heat conduction in fluid. The heat transfer rate is qualitatively described by the Landau equations for liquid He II with additional terms (Gorter-Mellink mutual friction) to account for frictional forces within the liquid. For the case of a cylindrical heater, the maximum steady state heat transfer rate before film boiling occurs depends on the depth of the heater below the liquid surface (hydrostatic head), the pressure of the gas above the liquid, and the liquid temperature. For a gas pressure of 20 Torr or more in excess of the equilibrium vapor pressure, the appearance of the film boiling changes from a uniform gas film to a fine haze, presumably of tiny gas bubbles. If He4 gas is used to pressurize the liquid, the temperature of the liquid He bath rises to about 2 K and a layer of liquid He I forms at the gas-liquid interface. This layer of the He I grows at the expense of the bulk He II as heat is supplied by the warm gas. The phase boundary between the two liquid phases moves down through the liquid at about 5 cm/min. The conditions for optimal heat transfer occur immediately after the pressure increase. These conditions are a pressure excess of 20 Torr or more and a bath temperature of 1.9 - 2 K.
Sporangiophores are suspended by an iron filing from an electromagnet in a special chamber (room temperature) above a helium cryostat. As soon as the pressure excess in the cryostat exceeds 20-60 Torr they are released and fall freely into the superfluid helium through a tube of heated gas. The sporangiophores are collected in a plastic beaker at the bottom of the cryostat and transferred to a liquid nitrogen storage dewar. Frozen sporangiophores are prepared for electron microscopy using freeze-substitution and freeze-fracture techniques.
The thin sections and the freeze-fracture replicas show extensive ice crystal damage to stage IV sporangiophores which have a large central vacuole. Ice crystal damage is considerably less in stage I sporangiophores which have a much smaller vacuole. Presumably, the vacuole is responsible for the poor preservation of the ultra-structure.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||Phycomyces liquid helium electron microscopy|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||17 August 1970|
|Author Email:||burke (AT) life.illinois.edu|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||08 Aug 2006|
|Last Modified:||21 Feb 2014 22:04|
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