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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

OsO4 1-4%
UA <3%
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:

Giddings, T.H. “Freeze Substitution protocols for improved visualization of membranes in high pressure frozen samples,” Journal of microscopy, vol 212 pt1 Oct 2003 pp 53-61 (discussion of FS solutions)

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.

Morphew-HPF-frz-subst-manual.pdf: Morphew, Mary K. "Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry," Boulder, CO

Steinbrecht and Muller, “Freeze Substitution and Freeze Drying,” Cryotechniques in Biological Electron Microscopy, chapter 7, Springer-Verlag, Berlin Heidelberg, 1987 (Solvent water capacity at low temp table)

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