Principles & Protocols

Principles - Freeze Substitution

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Important considerations in protocol design

• Rate of substitution process
  • faster substitution- larger ice crystal
  • slower substitution- discontinuous ice
• Efficiency of various organic solvents
• Sample size Solution volume= 1000x volume sample

Parameters:

• Temperature - determined by melting point of solvent

Solvents

• acetone- preserves membranes
• methanol- polarity such that it does not preserve membrane structure
• freezesub2.jpg:
  

Possible Fixatives

• Osmium tetroxide 1-4%
• Uranyl Acetate <3% - 0.1% prevents osmium from turning black
• Acrolien 5-20% in acetone Ornberg & Reese 1981 Murata 1985
• Glut <10%
• Hafnium chloride 0.5% in methanol

• Eutectic mixtures for freeze sub (Echlin 235)
• 70% ethylene glycol 30% H2O
• 40% ethylene glycol 50% Methanol

Example protocol

• Number 1
  • 72h @-90C
  • 12h@-60C
  • 6h@-30C
  • 3h@0C
  • 1h@room temperature (do not allow to sit at room temperature for more than one hour sample will turn black.)
  • All temperature changes with 5-10C/h slopes
  • Samples should be held at -90C for a minimum of 3 days
  • Warming samples to room temperature can take from 18h to 2 days
  • Substitution fluid can be changed during cycle
• Number 2
  • 72h @-90C
  • 12h@-30C
• Number 3
  • 8 h @-90C
  • 8 h @-60C
  • 8 h @-30C

Useful Reference Articles:

Kellenberger, Edward, “The response of Biological Macromolecules and Supramolecular Structures to the Physics of Specimen Cryoprepartation,” Cryotechniques in Biological Electron Microscopy, chapter 2, Springer-Verlag, Berlin Heidelberg, 1987 (hydration shell theory)

Mc Donald and Muller. "Exhibit C: Cryomethods for Thin Section Electron Microscopy." Sept 18, 2006.

Walther and Ziegler, “Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water,” J. Microscopy, vol 208 pt 1, pp 3-10, Oct 2002

Zalokar 1966 freeze sub rates with dye

Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup