Difference: CemProtocHPFU (1 vs. 27)

Revision 2729 Sep 2010 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Principles - High Pressure Freezing

Jump to Protocol

Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Liquid nitrogen cannot normally be used for freezing because of the Leidenfrost effect; the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. Increasing the pressure raises the vitreous freezing temperature. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen hydrated state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Pressure wave cracks samples

Filler selection - Looking for an osmotically inactive hydrophilic substance

Added:
>
>
  • Buffers are bad - salts crystalize
 

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
    • Vacuum infiltrate samples with hexadecane
    • Excess hexadecane not a problem will blow off in HPF
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Hat selection

  • Gold hats used for replicas because they will not dissolve in HCl
  • Water sticks to copper hats
  • Use stainless steal spacers between hats To mimic thickness of hat To use with sapphire disks to substitute hats

Solvent selection

  • Isopropanol is needed for freeze fracture only
  • Ethanol (technical) is okay for HPF

Loading

  • Use 1-2% agar as work surface. Prevents drying
Changed:
<
<
  • Lower hat with cells facing up
>
>
  • Work fast
Added:
>
>
  • Don't let cells dry out
  • Use right filler
  • Use smallest volume
 
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
Changed:
<
<
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
>
>
  • hat on top is dipped in hexadecene or set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 200 um without cryoprotectants
 
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
Changed:
<
<
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.
>
>
  • Hexadecene is a good lubricant to keep hats from sticking to the holder.
 

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 2602 Oct 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Principles - High Pressure Freezing

Jump to Protocol

Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Liquid nitrogen cannot normally be used for freezing because of the Leidenfrost effect; the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. Increasing the pressure raises the vitreous freezing temperature. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen hydrated state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
Added:
>
>

Pressure wave cracks samples

 

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
Added:
>
>
    • Vacuum infiltrate samples with hexadecane
    • Excess hexadecane not a problem will blow off in HPF
 
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter
Added:
>
>

Hat selection

  • Gold hats used for replicas because they will not dissolve in HCl
  • Water sticks to copper hats
  • Use stainless steal spacers between hats To mimic thickness of hat To use with sapphire disks to substitute hats

Solvent selection

  • Isopropanol is needed for freeze fracture only
  • Ethanol (technical) is okay for HPF
 

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 2503 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Principles - High Pressure Freezing

Jump to Protocol

Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Liquid nitrogen cannot normally be used for freezing because of the Leidenfrost effect; the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. Increasing the pressure raises the vitreous freezing temperature. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen hydrated state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 2402 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Principles - High Pressure Freezing

Jump to Protocol

Changed:
<
<		Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Increasing the pressure prevents the Leidenfrost effect, the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. The increase in pressure After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
>
>		Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Liquid nitrogen cannot normally be used for freezing because of the Leidenfrost effect; the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. Increasing the pressure raises the vitreous freezing temperature. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen hydrated state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
 

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 2302 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Changed:
<
<

High Pressure Freezing

>
>

Principles - High Pressure Freezing

 

Jump to Protocol

Deleted:
<
<

Principles

 Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Increasing the pressure prevents the Leidenfrost effect, the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. The increase in pressure After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 2202 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

High Pressure Freezing

Jump to Protocol

Principles

Changed:
<
<		Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
>
>		Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Increasing the pressure prevents the Leidenfrost effect, the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. The increase in pressure After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
 

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 2102 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

High Pressure Freezing

Jump to Protocol

Changed:
<
<

Princlples

>
>

Principles

 Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 2002 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Changed:
<
<

High Pressure Freezing and Freeze Substitution

>
>

High Pressure Freezing

 

Jump to Protocol

Changed:
<
<

Background

>
>

Princlples

 Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1902 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Changed:
<
<

High Pressure Freezing and Freeze Substitution

Jump to Protocol

>
>

High Pressure Freezing and Freeze Substitution

Jump to Protocol

 

Background

Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
Changed:
<
<

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

>
>

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

 
  • Glycerol
  • Ethylene glycol
Changed:
<
<

Non Penetrating Fillers

>
>

Non Penetrating Fillers

 
  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter
Changed:
<
<

Loading

>
>

Loading

 
  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.
Changed:
<
<

Embedding

>
>

Embedding

 
  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.
Changed:
<
<

Substitution

>
>

Substitution

 
  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each
Changed:
<
<

Protocol for yeast

>
>

Protocol for yeast

 
  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Changed:
<
<

Protocols

>
>

Protocols

 

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1802 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Added:
>
>		Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

 

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1702 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
Changed:
<
<
  • Try using 20% BSA as a filler. Maybe more reproducible water retention properties than dextran.
    • Yeast comes from Drosophila food.
>
>
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter
Deleted:
<
<
    • Bakers yeast ok also. Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter (Kent's favorite)
 

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

Changed:
<
<
>
>
  • en face.
Deleted:
<
<
  • Kent uses Epon/Araldite mix, but pure EPON should be OK.
  • Kent generally cuts en face.
 
    • Could use flat molds and place hats at the tip and try to cut in cross section.
Changed:
<
<
    • Devrim prefers reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
>
>
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
Added:
>
>
  • sapphire disk tricks
 
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

Deleted:
<
<
 
  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

Deleted:
<
<
 
  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1602 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
Added:
>
>
  • Try using 20% BSA as a filler. Maybe more reproducible water retention properties than dextran.
    • Yeast comes from Drosophila food.
    • Bakers yeast ok also. Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter (Kent's favorite)
 

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.
Deleted:
<
<
  • Fillers
    • Try using 20% BSA as a filler. Maybe more reproducible water retention properties than dextran.
    • Yeast comes from Drosophila food.
    • Bakers yeast ok also. Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter (Kent's favorite)
    • Hexadecene
 

Embedding

  • Kent uses Epon/Araldite mix, but pure EPON should be OK.
  • Kent generally cuts en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • Devrim prefers reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1502 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

Changed:
<
<		(in progress)
>
>		Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants under normal atmospheric conditions. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
Deleted:
<
<		Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants in normal atmospheric conditions. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that extract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
 

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

  • Fillers
    • Try using 20% BSA as a filler. Maybe more reproducible water retention properties than dextran.
    • Yeast comes from Drosophila food.
    • Bakers yeast ok also. Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter (Kent's favorite)
    • Hexadecene

Embedding

  • Kent uses Epon/Araldite mix, but pure EPON should be OK.
  • Kent generally cuts en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • Devrim prefers reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1402 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

(in progress)
Changed:
<
<		Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
>
>		Biological specimens thicker than 250 nm are too thick to form vitreous ice without the use of cryo protectants in normal atmospheric conditions. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that extract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
 

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)

Loading

Deleted:
<
<
 
  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

  • Fillers
    • Try using 20% BSA as a filler. Maybe more reproducible water retention properties than dextran.
    • Yeast comes from Drosophila food.
    • Bakers yeast ok also. Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter (Kent's favorite)
    • Hexadecene

Embedding

  • Kent uses Epon/Araldite mix, but pure EPON should be OK.
  • Kent generally cuts en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • Devrim prefers reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1302 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

  • Fillers
    • Try using 20% BSA as a filler. Maybe more reproducible water retention properties than dextran.
    • Yeast comes from Drosophila food.
    • Bakers yeast ok also. Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter (Kent's favorite)
    • Hexadecene
Added:
>
>

Embedding

  • Kent uses Epon/Araldite mix, but pure EPON should be OK.
  • Kent generally cuts en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • Devrim prefers reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.
 

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1202 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
Changed:
<
<

Loading

>
>

Loading

Added:
>
>
 
  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
Added:
>
>
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

  • Fillers
    • Try using 20% BSA as a filler. Maybe more reproducible water retention properties than dextran.
    • Yeast comes from Drosophila food.
    • Bakers yeast ok also. Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter (Kent's favorite)
    • Hexadecene
 

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1102 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Jump to Protocol

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
Changed:
<
<

Protocols

>
>

Loading

Added:
>
>
  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
 
Changed:
<
<
>
>

Protocols

 

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 1002 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"

High Pressure Freezing and Freeze Substitution

Changed:
<
<		 Jump to Protocol
>
>

Jump to Protocol

 

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 902 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Deleted:
<
<		Contents
 

High Pressure Freezing and Freeze Substitution

Changed:
<
<
>
>		 Jump to Protocol
 

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 802 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Added:
>
>
 

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)

Protocols

Changed:
<
<
>
>
 

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 726 Aug 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.
Added:
>
>

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
 

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 610 Aug 2009 - Main.BillRice

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Protocols

Added:
>
>
 
  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 510 Aug 2009 - Main.BillRice

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Protocols

Changed:
<
<		HighPressFreezer FreezeSubstitution
>
>
 
  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 410 Aug 2009 - Main.BillRice

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Protocols

Added:
>
>		HighPressFreezer
 FreezeSubstitution

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 310 Aug 2009 - Main.BillRice

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Background

(in progress) Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Protocols

Added:
>
>		FreezeSubstitution
 
  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 210 Aug 2009 - Main.BillRice

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Changed:
<
<

Sublevel topic

subsub level topic

>
>

Background

(in progress)
Added:
>
>		Biological specimens, such as tissue, cultured cells, or organelles, which are thicker than 250 nm must be sectioned prior to electron microscopy. The challenge is to preserve the structural integrity of macromolecular complexes within these sections. Conventional techniques employ chemical fixation, staining, dehydration and embedding in polymer resins prior to cutting sections with an ultramicrotome. The conventional protocols involve harsh treatments that exract substantial biological materials and fail to preserve details finer than ~20 nm. Cryogenic methods offer substantially better preservation. Although plunge-freezing is limited to samples <5-10 micrometers in thickness, high-pressure freezing is suitable for samples up to 100 micrometers in thickness. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen, unstained state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Protocols

 
  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

Revision 110 Aug 2009 - Main.BillRice

 
META TOPICPARENT name="CemProtoc"
Contents

High Pressure Freezing and Freeze Substitution

Sublevel topic

subsub level topic

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

View topic  |  History: r27 < r26 < r25 < r24  |  More topic actions...
 
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