TITLE: Preparation of cells for transmission electron microscopy ultrastructural analysis
AUTHORS: Jillian  C Danne, Simon A Crawford, Rachel Templin, Joan Clark, Viola Oorschot, Georg  Ramm

[DESCRIPTION]
A transmission electron microscope (TEM) enables the magnification and visualisation of cell and tissue ultrastructure that cannot otherwise be resolved using a light microscope. In transmission electron microscopy, a high energy electron beam generated from an electron gun is focused by electromagnetic lenses onto an ultrathin section of plastic embedded specimen. Electrons are transmitted through the specimen onto a fluorescent screen based on sample density, and an image acquired by a digital camera. Here, a protocol for preparing cells for TEM ultrastructural analysis is described. A cell sample is fixed, dehydrated and infiltrated with resin. The resin is then polymerised, and embedded cell sample sectioned using an ultramicrotome. Ultrathin sections are contrasted with heavy metals prior to imaging using a TEM. Options for faster processing using a Microwave processor are described for relevant steps.

[STEPS]
SECTION: Fixation
1. All fixation and processing steps must be performed in a fume hood wearing the appropriate personal protective equipment (PPE). The Material Safety Data Sheet (MSDS) for each chemical must be read before commencing.

Adherent cells grown in a well-plate or petri dish must be submerged in solution at all times to prevent desiccation. 

Cells suspended in culture media in 1.5 ml microcentrifuge or 15 ml Falcon® tubes require centrifuging at 1200 rpm for 2 minutes prior to fixation and following Steps 1-4. After removal of the supernatant, cell pellets should be gently resuspended in solution using a pipette.


Adherent cells:
Remove cell culture media from the well-plate or petri dish and gently add Karnovsky's fixative, 2% PFA, 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). Seal the well-plate or petri dish lid with parafilm, and fix the cells for 2 hours at room temperature or overnight at 4 degrees Celsius, agitating. 

Suspended cells:
Remove cell culture media from the 1.5 ml microcentrifuge or 15 ml Falcon® tube using a pipette and gently resuspend the cell pellet in 1 ml of Karnovsky's fixative, 2% PFA, 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). Fix cells for 2 hours at room temperature or overnight at 4 degrees Celsius, agitating. 

SECTION: Fixation
2. Remove fixative and wash with 0.1M cacodylate buffer, 3 x 10 minutes at room temperature, agitating. 
SECTION: Dehydration and resin infiltration
6. 
The following steps can be performed with or without the use of a biowave. A biowave facilitates infiltration of reagents into tissue and cells, and reduces the processing time: 

For Steps 7-20, do not proceed if samples float following ethanol, propylene oxide or Epon resin infiltration. 
Well-infiltrated samples should sink in the tube. If they do not, samples may require extended infiltration times. 

Epon resin can be stored in the freezer but must come to room temperature before use. 


Dehydrate in 30% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
8. Dehydrate in 70% ethanol in MilliQ water for 1 hour at room temperature, agitating. Tissue can be left at 4 degrees, overnight if necessary.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
9. Dehydrate in 90% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
10. Dehydrate in 95% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
11. Dehydrate in 100% ethanol for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
12. Dehydrate again in 100% ethanol for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
13. Infiltrate with propylene oxide for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
15. Infiltrate with propylene oxide : epon resin (3 : 1 ratio) overnight at room temperature, agitating. 

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
17. Infiltrate with propylene oxide : epon resin (1 : 3 ratio) overnight at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on. 
SECTION: Dehydration and resin infiltration
18. Infiltrate with 100 % epon resin for 6 hours at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
19. Infiltrate with 100 % epon resin overnight at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
20. Transfer samples to plastic embedding moulds or flat bottom Beem capsules containing 100% epon resin. Polymerise in an oven set to 60 degrees Celsius for 24-48 hours. 
SECTION: Dehydration and resin infiltration
16. Infiltrate with propylene oxide : epon resin (1 : 1 ratio) for 6 hours at room temperature, agitating. 

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Fixation
3. Osmium tetroxide is extremely toxic and must be handled with care in a fume hood using the appropriate PPE. Used osmium should be discarded in a labelled plastic container containing ethanol to reduce and neutralise the osmium.

Osmium can precipitate out of solution when in the presence of glutaraldehyde to form small electron dense artifactual deposits. Samples must be washed thoroughly (Step 2) prior to post-fixation (Step 3) to avoid this.

Potassium ferrocyanide reduces osmium, turning the post fixation solution black. Reduced osmium may also prevent the formation of artefactual precipitates. Potassium ferricyanide can be used as an alternative in combination with osmium tetroxide. This solution is a clear, amber colour.


Post fix cells in 1% Osmium tetroxide, 1.5% potassium ferrocyanide in 0.1M cacodylate buffer for 1 hour at room temperature, agitating. Well-plate, petri dish and tube lids must be well-secured during fixation. 


SECTION: Dehydration and resin infiltration
14. Infiltrate again with propylene oxide for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
7. Dehydrate in 50% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Semi and ultrathin sectioning
21. Mount the resin-embedded tissue block in an ultramicrotome chuck, and secure the chuck to a stereo microscope fitted with a chuck mount. Manually trim the front face and all four edges of the tissue block into a trapezoid shape using a double edge razor blade. 
SECTION: Semi and ultrathin sectioning
24. Once the region of interest has been obtained, cut 90 nm gold coloured ultrathin sections at 1mm/sec using a Leica UC7 ultramicrotome and a Diatome 45 degree angle ultra wet diamond knife.
SECTION: Semi and ultrathin sectioning
25. Pick up sections on 50-200 mesh copper or palladium-coated copper grids. Place grids section side up on a sheet of filter paper and leave to dry for 10 minutes. Store grids in an enclosed grid box until use.
SECTION: Semi and ultrathin sectioning
23. To check the region of interest:  
SECTION: Semi and ultrathin sectioning
23.1. Cut 500nm semi thin sections at a speed of 1mm/sec using a Leica UC7 ultramicrotome and 45 degree glass knife fitted with a water-filled boat. 
SECTION: Semi and ultrathin sectioning
23.2. Retrieve sections with an eyelash or Dalmatian hair mounted on a wooden stick and float sections in a drop of MilliQ water on a slide before drying on a mini hot plate. 
SECTION: Semi and ultrathin sectioning
23.3. Stain sections with toluidine blue solution for 10-20 seconds before rinsing with MilliQ water and drying on a mini hot plate.

Toluidine blue stains nucleic acids and proteins, enhancing structural detail of tissue semi thin sections.
SECTION: Semi and ultrathin sectioning
23.4. Observe sections with a wide-field light microscope. If the region of interest has not been obtained, trim deeper and repeat Step 24. 
SECTION: Semi and ultrathin sectioning
22. Transfer the chuck and resin-embedded tissue block to a Leica UC7 ultramicrotome and secure in place. Trim the front surface of the block face until a full tissue face has been obtained. Cut at a speed of 1mm/sec using a 45 degree glass knife (thickness 6.4 mm or 8 mm). 
SECTION: Contrasting ultrathin sections
26. Place a sheet of parafilm on a clean flat bench or a glass board using a small amount of water underneath to keep the film flat. 

For the following steps, use fine forceps or a perfect loop to transfer grids from one drop of solution to another, section side down. Use approximately 100-200 μl drops for MilliQ water, and 50-100 μl for uranyl acetate and lead citrate.
SECTION: Contrasting ultrathin sections
27. Stain sections with 0.6% filtered uranyl acetate in MilliQ water for 3 minutes.

Uranyl acetate enhances contrast of ultrathin tissue sections by interacting with lipids and proteins. The solution must be wrapped in foil and stored in a dark place to prevent precipitation from exposure to UV light.
SECTION: Contrasting ultrathin sections
28. Rinse 5 x 1 min in MilliQ water.
SECTION: Contrasting ultrathin sections
29. Stain sections with 0.1-0.4% filtered lead citrate in MilliQ water for 3 minutes. 

Four drops of 10M sodium hydroxide should be added to the lead citrate stock solution prior to use to prevent lead citrate precipitating to form lead carbonate by exposure to carbon dioxide. 

Lead citrate enhances contrast of ultrathin tissue sections by interacting with proteins and glycogens.
SECTION: Contrasting ultrathin sections
30. Rinse 5 x 1 min in MilliQ water.
SECTION: Contrasting ultrathin sections
31. Place grids section side up on a sheet of filter paper to dry for 10 minutes and store in a grid storage box.
SECTION: Contrasting ultrathin sections
32. Place the grid in a transmission electron microscope grid holder for high resolution imaging using a transmission electron microscope.

Example: JEOL 1400-Plus 120 keV TEM equipped with a high sensitivity bottom mount CMOS 'Flash' camera.
SECTION: Fixation
5. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Washed samples can be stored at 4 degrees Celsius for up to 1 week until further processing.


SECTION: Fixation
4. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Washed samples can be stored at 4 degrees Celsius for up to 1 week until further processing. If handling suspended cells, proceed to Step 6.

SECTION: Fixation
4. 
SECTION: Agarose embedding
7. 
Melt 4% aqueous ultra-pure agarose using a Bain Marie water bath containing boiling water or a heat block set to 95 degrees Celsius. Ultra-pure agarose solidifies at ≤ 32.5 - 38 degrees Celsius and melts at ≥ 90 degrees Celsius. 

SECTION: Agarose embedding
8. Remove the supernatant and add approximately 100 μl (2 drops) of the pre-warmed 4% aqueous agarose to the cell pellet.
SECTION: Agarose embedding
9. Gently vortex the sample to homogeneously resuspend the cell pellet in pre-warmed 4% aqueous agarose.
SECTION: Agarose embedding
10. Optionally, to obtain a concentrated cell pellet, return the microcentrifuge tube containing cells in 4% aqueous agarose to the Bain Marie or heat block until the agarose liquefies, and then pellet cells using a benchtop microcentrifuge.




SECTION: Agarose embedding
11. Solidify the 4% aqueous agarose with cells by cooling at 4 degrees Celsius for 10 minutes.  


SECTION: Agarose embedding
12. Cut off the extreme tip of the microcentrifuge tube (avoiding the cells) using a razor blade and push the sample out of the tube using a wooden stick.
SECTION: Agarose embedding
13. Place the agarose embedded cell block on a Teflon plate or dental wax sheet and cut the block in half using a scalpel or double edged razor blade. Cut each block half into strips and then cut each strip into cubes no larger than 1mm3. Carefully place the agarose-embedded cell cubes into a 5ml tube using forceps.



SECTION: Dehydration and resin infiltration
14. For Steps 14-27, do not proceed if samples float following ethanol, acetone or Epon resin infiltration. 
Well-infiltrated samples should sink in solution. If they do not, samples may require extended infiltration times. 

Epon resin can be stored in the freezer but must come to room temperature before use. 

The following steps can be performed with or without the use of a PELCO BioWave® Pro+ Microwave Processing System. A Biowave facilitates infiltration of reagents into tissue and cells, and reduces the processing time.


Dehydrate in 30% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
15. Dehydrate in 50% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
16. Dehydrate in 70% ethanol in MilliQ water for 1 hour at room temperature, agitating. The sample can be left at 4 degrees Celsius, overnight if necessary.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
17. Dehydrate in 90% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
18. Dehydrate in 95% ethanol in MilliQ water for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
19. Dehydrate in 100% ethanol for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
20. Dehydrate in 100% anhydrous ethanol for 1 hour at room temperature, agitating. Type 3Å molecular sieves can be added to the ethanol stock solution to dehydrate the solvent prior to use. 

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
21. Dehydrate in 100% acetone for 1 hour at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum.

Propylene oxide can be used as an alternative to acetone (Steps 21-25), but is extremely toxic and may dehydrate samples too intensively, causing ultrastructural damage. 
SECTION: Dehydration and resin infiltration
22. Dehydrate in 100% anhydrous acetone for 1 hour at room temperature, agitating. Type 3Å molecular sieves can be added to the acetone stock solution to dehydrate the solvent prior to use. 

Biowave settings: 40 seconds, 150W, No vacuum.
SECTION: Dehydration and resin infiltration
23. Optionally Infiltrate with 100% anhydrous acetone : epon resin (3 : 1 ratio) overnight at room temperature, agitating. 

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
24. Infiltrate with 100% anhydrous acetone : epon resin (1 : 1 ratio) for 6 hours at room temperature, agitating. 

Biowave settings: 3 minutes, 250W, Vacuum on.
SECTION: Dehydration and resin infiltration
25. Infiltrate with 100% anhydrous acetone : epon resin (1 : 3 ratio) overnight at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on. 
SECTION: Dehydration and resin infiltration
26. Infiltrate with 100% epon resin for 6 hours at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on.


SECTION: Dehydration and resin infiltration
27. Infiltrate with 100% epon resin overnight at room temperature, agitating.

Biowave settings: 3 minutes, 250W, Vacuum on.

Residual acetone can adversely impact resin infiltration and polymerisation. A third 100% resin infiltration step may  be required to prevent this.
SECTION: Dehydration and resin infiltration
28. Transfer samples to labelled plastic embedding moulds, flat bottom BEEM® capsules or standard tip BEEM® capsules containing 100% epon resin. Polymerise in an oven set to 60 degrees Celsius for 24-48 hours. 
SECTION: Semi and ultrathin sectioning
29. Mount the resin-embedded cell block in an ultramicrotome chuck, and secure the chuck to a stereo microscope fitted with a chuck mount. Manually trim the front face and all four edges of the cell block into a trapezoid shape using a double edge razor blade. 
SECTION: Semi and ultrathin sectioning
30. Transfer the chuck and resin-embedded cell block to a Leica UC7 ultramicrotome and secure in place. Trim the front surface of the block face until a full face of cells has been obtained. Trim manually using a 0.5 μm feed and a 45 degree glass knife (thickness 6.4 mm or 8 mm) or suitable diamond knife, for example a Diatome 35 degree angle ultra semi diamond knife.
SECTION: Semi and ultrathin sectioning
31. To check the region of interest:  
SECTION: Semi and ultrathin sectioning
32. Cut 90 nm gold coloured ultrathin sections at 1 mm/sec using a Leica UC7 ultramicrotome and a Diatome 45 degree angle ultra wet diamond knife. Spread the sections using a Max Wax heat pen to prevent wrinkles. 
SECTION: Semi and ultrathin sectioning
33. Pick up sections on 50-200 mesh copper or palladium-coated copper grids. Place grids section side up on a sheet of filter paper, cover with a glass petri dish and leave to dry for 10 minutes. Store grids in an enclosed grid box until use.
SECTION: Semi and ultrathin sectioning
31.1. Cut 500 nm semi thin sections at a speed of 1 mm/sec using a Leica UC7 ultramicrotome and 45 degree glass knife fitted with a filtered water-filled boat or suitable diamond knife.  
SECTION: Semi and ultrathin sectioning
31.2. Retrieve sections with an eyelash or Dalmatian hair mounted on a wooden stick and float sections in a drop of MilliQ water on a slide before drying on a mini hot plate. 
SECTION: Semi and ultrathin sectioning
31.3. Stain sections with Toluidine blue solution for 10-20 seconds on a mini hot plate before rinsing with MilliQ water and drying on a mini hot plate.

Toluidine blue stains nucleic acids and proteins, enhancing structural detail of semi thin cell sections.
SECTION: Semi and ultrathin sectioning
31.4. Observe sections with a wide-field light microscope. If the region of interest has not been obtained, trim deeper and repeat Step 31. 
SECTION: Contrasting ultrathin sections
34. Place a sheet of parafilm on a clean flat bench or glass board using a small amount of water underneath to keep the film flat. 

For the following steps, use fine forceps or a perfect loop to transfer grids from one drop of solution to another, section side down. Use approximately 100-200 μl drops for MilliQ water, and 50-100 μl for uranyl acetate and lead citrate.
SECTION: Contrasting ultrathin sections
35. Stain sections with 0.6% filtered uranyl acetate in MilliQ water for 3 minutes.

Uranyl acetate enhances contrast of sections by interacting with proteins and lipids. The solution must be wrapped in foil and stored in a dark place to prevent precipitation from exposure to UV light. 
SECTION: Contrasting ultrathin sections
36. Rinse 5 x 1 min in MilliQ water.
SECTION: Contrasting ultrathin sections
37. Stain sections with 0.1-0.4% filtered lead citrate in MilliQ water for 3 minutes. 

Four drops of 10M sodium hydroxide should be added to the lead citrate stock solution prior to use to prevent lead citrate precipitating to form water-insoluble lead carbonate by exposure to carbon dioxide. 

Lead citrate enhances contrast of sections by interacting with proteins and glycogens, and binding to osmium and uranyl acetate.
SECTION: Contrasting ultrathin sections
38. Rinse 5 x 1 min in MilliQ water.
SECTION: Contrasting ultrathin sections
39. Place grids section side up on a sheet of filter paper, cover with a glass petri dish and dry for 10 minutes. Store grids in a grid storage box.
SECTION: Contrasting ultrathin sections
40. Place a grid in a transmission electron microscope grid holder for high resolution imaging using a transmission electron microscope.

Example: JEOL JEM-1400-Plus TEM at 80 keV equipped with a high sensitivity bottom mount CMOS 'Flash' camera.
SECTION: Fixation
5. Gently detach adherent cells from the bottom of the well-plate/petri dish using a cell scraper. 
SECTION: Fixation
6. Transfer cells to a 1.5 ml microcentrifuge tube using a 3 ml or 1000 μl transfer pipette, and pellet cells at 1200 rpm for 2 minutes using a benchtop centrifuge.