Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

• 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.

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

• 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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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 * freezesub.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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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 * freezesub.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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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 * freezesub.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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

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:

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

Possible Fixatives

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.

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

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

Possible Fixatives

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.

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

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

Important considerations in protocol design

• Rate of substitution process

• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

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

• 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

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Example protocols

• 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

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Example protocols

• 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

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Jump to Protocol

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Principles - Freeze Substitution

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

Solution Prepartation

Here is Kent Mc Donald's recipe for preparing FS solutions (from Exhibit C):

1. Label, with the #2 pencil, about 35 Nalgene 2.0 ml cryovials.
2. Put vials in vial rack with tops off.
3. Make up stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 ml pure methanol, cover with foil and put on rocker to dissolve. It should dissolve in about 10 minutes, but if it doesn't, just let it rock longer. Different vintages of uranyl acetate crystals seem to dissolve at different rates.
4. Dip a 1 g OsO4^? ampule for about 10 seconds into liquid nitrogen to loosen crystals from glass.
5. Fill 50 ml conical tube with 45 ml pure acetone.
6. Add 1 g OsO4^? crystals to the 45 ml of acetone, cover and mix until completely dissolved.
7. Add 1 ml 5% uranyl acetate stock to OsO4^?-acetone, add pure acetone to make 50 ml and mix well.
8. Use repeater pipettor to dispense 1.5 ml into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
9. Add LN2 to Styrafoam box to a level that will cover cryovials in rack.
10. Slowly lower rack into LN2. Vials must remain upright so fixative will remain in the bottom of the vial.
11. When completely frozen, transfer vials to LN2 storage device until ready to use.

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Freeze Substitution Guidelines

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

Solution Prepartation

Here is Kent Mc Donald's recipe for preparing FS solutions (from Exhibit C):

1. Label, with the #2 pencil, about 35 Nalgene 2.0 ml cryovials.
2. Put vials in vial rack with tops off.
3. Make up stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 ml pure methanol, cover with foil and put on rocker to dissolve. It should dissolve in about 10 minutes, but if it doesn't, just let it rock longer. Different vintages of uranyl acetate crystals seem to dissolve at different rates.
4. Dip a 1 g OsO4^? ampule for about 10 seconds into liquid nitrogen to loosen crystals from glass.
5. Fill 50 ml conical tube with 45 ml pure acetone.
6. Add 1 g OsO4^? crystals to the 45 ml of acetone, cover and mix until completely dissolved.
7. Add 1 ml 5% uranyl acetate stock to OsO4^?-acetone, add pure acetone to make 50 ml and mix well.
8. Use repeater pipettor to dispense 1.5 ml into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
9. Add LN2 to Styrafoam box to a level that will cover cryovials in rack.
10. Slowly lower rack into LN2. Vials must remain upright so fixative will remain in the bottom of the vial.
11. When completely frozen, transfer vials to LN2 storage device until ready to use.

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Freeze Substitution Guidelines

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Freeze Substitution

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

Solution Prepartation

Here is Kent Mc Donald's recipe for preparing FS solutions (from Exhibit C):

1. Label, with the #2 pencil, about 35 Nalgene 2.0 ml cryovials.
2. Put vials in vial rack with tops off.
3. Make up stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 ml pure methanol, cover with foil and put on rocker to dissolve. It should dissolve in about 10 minutes, but if it doesn't, just let it rock longer. Different vintages of uranyl acetate crystals seem to dissolve at different rates.
4. Dip a 1 g OsO4^? ampule for about 10 seconds into liquid nitrogen to loosen crystals from glass.
5. Fill 50 ml conical tube with 45 ml pure acetone.
6. Add 1 g OsO4^? crystals to the 45 ml of acetone, cover and mix until completely dissolved.
7. Add 1 ml 5% uranyl acetate stock to OsO4^?-acetone, add pure acetone to make 50 ml and mix well.
8. Use repeater pipettor to dispense 1.5 ml into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
9. Add LN2 to Styrafoam box to a level that will cover cryovials in rack.
10. Slowly lower rack into LN2. Vials must remain upright so fixative will remain in the bottom of the vial.
11. When completely frozen, transfer vials to LN2 storage device until ready to use.

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Freeze Substitution Guidelines

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Freeze Substitution

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

Solution Prepartation

Here is Kent Mc Donald's recipe for preparing FS solutions (from Exhibit C):

1. Label, with the #2 pencil, about 35 Nalgene 2.0 ml cryovials.
2. Put vials in vial rack with tops off.
3. Make up stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 ml pure methanol, cover with foil and put on rocker to dissolve. It should dissolve in about 10 minutes, but if it doesn't, just let it rock longer. Different vintages of uranyl acetate crystals seem to dissolve at different rates.
4. Dip a 1 g OsO4^? ampule for about 10 seconds into liquid nitrogen to loosen crystals from glass.
5. Fill 50 ml conical tube with 45 ml pure acetone.
6. Add 1 g OsO4^? crystals to the 45 ml of acetone, cover and mix until completely dissolved.
7. Add 1 ml 5% uranyl acetate stock to OsO4^?-acetone, add pure acetone to make 50 ml and mix well.
8. Use repeater pipettor to dispense 1.5 ml into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
9. Add LN2 to Styrafoam box to a level that will cover cryovials in rack.
10. Slowly lower rack into LN2. Vials must remain upright so fixative will remain in the bottom of the vial.
11. When completely frozen, transfer vials to LN2 storage device until ready to use.

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Freeze Substitution Guidelines

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Freeze Substitution

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

Solution Prepartation

Here is Kent Mc Donald's recipe for preparing FS solutions (from Exhibit C):

1. Label, with the #2 pencil, about 35 Nalgene 2.0 ml cryovials.
2. Put vials in vial rack with tops off.
3. Make up stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 ml pure methanol, cover with foil and put on rocker to dissolve. It should dissolve in about 10 minutes, but if it doesn't, just let it rock longer. Different vintages of uranyl acetate crystals seem to dissolve at different rates.
4. Dip a 1 g OsO4^? ampule for about 10 seconds into liquid nitrogen to loosen crystals from glass.
5. Fill 50 ml conical tube with 45 ml pure acetone.
6. Add 1 g OsO4^? crystals to the 45 ml of acetone, cover and mix until completely dissolved.
7. Add 1 ml 5% uranyl acetate stock to OsO4^?-acetone, add pure acetone to make 50 ml and mix well.
8. Use repeater pipettor to dispense 1.5 ml into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
9. Add LN2 to Styrafoam box to a level that will cover cryovials in rack.
10. Slowly lower rack into LN2. Vials must remain upright so fixative will remain in the bottom of the vial.
11. When completely frozen, transfer vials to LN2 storage device until ready to use.

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Principles & Protocols

Important consideration in protocol design

• Rate of substitution process
• Efficiency of various organic solvents

Parameters:

• Temperature - determined by melting point of solvent

Solution Prepartation

Here is Kent Mc Donald's recipe for preparing FS solutions (from Exhibit C):

1. Label, with the #2 pencil, about 35 Nalgene 2.0 ml cryovials.
2. Put vials in vial rack with tops off.
3. Make up stock solution of 5% uranyl acetate in methanol by adding 0.1 g of uranyl acetate to 2 ml pure methanol, cover with foil and put on rocker to dissolve. It should dissolve in about 10 minutes, but if it doesn't, just let it rock longer. Different vintages of uranyl acetate crystals seem to dissolve at different rates.
4. Dip a 1 g OsO4^? ampule for about 10 seconds into liquid nitrogen to loosen crystals from glass.
5. Fill 50 ml conical tube with 45 ml pure acetone.
6. Add 1 g OsO4^? crystals to the 45 ml of acetone, cover and mix until completely dissolved.
7. Add 1 ml 5% uranyl acetate stock to OsO4^?-acetone, add pure acetone to make 50 ml and mix well.
8. Use repeater pipettor to dispense 1.5 ml into each cryovial. Work quickly, but not carelessly. Cap cryovials as soon as they are filled.
9. Add LN2 to Styrafoam box to a level that will cover cryovials in rack.
10. Slowly lower rack into LN2. Vials must remain upright so fixative will remain in the bottom of the vial.
11. When completely frozen, transfer vials to LN2 storage device until ready to use.

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, Mary K. “Practical Methods in High Pressure Freezing, Freeze Substitution, Embedding and Immunocytochemistry,” Can Download from specimen preparation page of: http://bio3d.colorado.edu/ (good general and some FS solution explanation)

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

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)
• Embedding_Schedule_for_HPF_Samples.doc:

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• Large forceps
• Styrofoam box to work in
• Pipettes
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil

• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)
• Transfer frozen hats to frozen substitution solution under liquid nitrogen environment
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare substituion solution (e.g. OsO4 solution^?)

• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil

• Transfer frozen hats to frozen substitution solution in liquid nitrogen
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• Pencil
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil

• Transfer frozen hats to frozen substitution solution in liquid nitrogen
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Gloves
• Oxygen Moniter
• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol

• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS through funnel with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent methanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Prepare SubstituionSolution
• Transfer frozen hats to frozen substitution solution in liquid nitrogen
• Place maximum of two specimen per cryotube
• Insert samples into AFS sample well
• Begin freeze substitution cycle

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Freeze Substitution Guidelines

Contents

You will need:

• Cryovials
• Substitution fluid (e.g. OsO4 solution^?)
• Methanol
• Ethanol
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent ethanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Transfer frozen hats to frozen acetone solution in liquid nitrogen
• Place max of two specimen per cryotube
• Insert samples into sample well
• Begin freeze substitution

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

Freeze Substitution Guidelines

Contents

You will need:

• Cryovials

• Methanol
• Ethanol
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent ethanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Transfer frozen hats to frozen acetone solution in liquid nitrogen
• Place max of two specimen per cryotube
• Insert samples into sample well
• Begin freeze substitution

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

Freeze Substitution Guidelines

Contents

You will need:

• Cryovials
• Substitution fluid
• Methanol
• Ethanol
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent ethanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Transfer frozen hats to frozen acetone solution in liquid nitrogen
• Place max of two specimen per cryotube
• Insert samples into sample well
• Begin freeze substitution

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005

Freeze Substitution Guidelines

Contents

You will need:

• Cryovials
• Substitution fluid
• Methanol
• Ethanol
• LN2 (tank full)

Procedure:

• Wear oxygen monitor and fill AFS with liquid nitrogen.
• Warm up sample well to 30C for 30 min to remove all moisture.

• After heat cycle fill sample well with reagent ethanol
• Program substitution sequence
  • If a sequence is running press pause and hold until you hear three beeps
• Begin program and pause until starting temperature is reached

• Label Nalgene cryotubes with pencil
• Transfer frozen hats to frozen acetone solution in liquid nitrogen
• Place max of two specimen per cryotube
• Insert samples into sample well
• Begin freeze substitution

Example protocols

• 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

Finally

• Rinse 3x with dehydrated acetone for 1h each
• Infiltrate with resin

References

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

-- DavidStokes - 18 Mar 2005