TITLE: Preparation of Cells for Volume Electron Microscopy using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM)
AUTHORS: Jillian C Danne, Rachel Templin, Gediminas Gervinskas, Denis Korneev, Sergey Gorelick, Georg Ramm

[DESCRIPTION]
Volume Electron Microscopy (vEM) allows for the three-dimensional imaging of biological matter and to observe cellular structures at the nanometer scale. This protocol details the preparation of cellular specimens for Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). FIB-SEM gives the highest resolution in z direction compared to other vEM techniques. Images are obtained by stepwise slicing of a specimen using the ion beam while imaging successive surfaces by scanning electron microscopy. Good structural preservation and electron contrast are essential, and this protocol details the required preparatory steps for this up to (but not including) the slice and view imaging in the FIB-SEM. 

[STEPS]
SECTION: Fixation
1. All fixation and processing steps must be performed in a fume hood wearing 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. Adherent cells can either be scraped and then processed as an agarose embedded block, or kept intact and processed as a single cell monolayer. 

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 1-13. 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% paraformaldehyde (PFA), 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4). 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. 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: 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 artefactual deposits. Samples must be washed thoroughly (Step 2) prior to post-fixation (Step 3) to avoid this. 

The following Steps 3 - 13 and Steps 24-38 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. 

Post fix cells in 2% osmium tetroxide in 0.1M cacodylate buffer for 30 mins at room temperature, agitating. Well-plate, petri dish and tube lids must be well-secured during fixation. 

The microwave regime described below (Steps 3, 4, 6, 8, 10, 12) cycles between an on and off step, repeating 3 times.  The microwave off step prevents overheating of the sample. 

Biowave settings: Repeat cycle 3 times:
2 mins Microwave 100W, Vacuum on, at room temperature
2 mins Microwave off, Vacuum on, at room temperature

Osmium tetroxide stains and fixes lipids by oxidising unsaturated fatty acid bonds. It penetrates deeper into a sample when it is not reduced in solution by potassium ferrocyanide or potassium ferricyanide. 
SECTION: Fixation
4. Post fix the cells in 1.5% potassium ferrocyanide in 0.1M cacodylate buffer for 30 mins at room temperature, agitating. Tube and well-plate lids must be well-secured during fixation. 

Biowave settings: Repeat cycle 3 times:
2 mins Microwave 100W, Vacuum on, at room temperature
2 mins Microwave off, Vacuum on, at room temperature

Potassium ferrocyanide is a reducing agent that improves contrast of biological samples by enhancing the amount of electron dense osmium deposited in membranes. When used in combination with osmium, osmium becomes reduced and the post fixation solution turns dark brown. Reduced osmium improves membrane and glycogen contrast with limited penetration, and may also prevent the formation of artifactual precipitates. Potassium ferricyanide can be used as an alternative to potassium ferrocyanide and remains a clear, amber colour if mixed with osmium. 
SECTION: Fixation
5. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Biowave settings: 3 x 40 seconds, 150W, Vacuum off, at room temperature.  
SECTION: Fixation
6. Incubate cells in 1% Thiocarbohydrazide in MilliQ water for 20 mins at 40 degrees Celsius, agitating. Tube and well-plate lids must be well-secured during this step. 

Biowave settings: Repeat cycle 3 times:
2 mins Microwave 150W, Vacuum on, at 40 degrees Celsius
2 mins Microwave off, Vacuum on, at 40 degrees Celsius

Thiocarbohydrazide binds to osmium and acts as a bridge for the binding of additional osmium in Step 8, thereby enhancing contrast of lipid membranes. The increase in osmium content also makes cells more conductive.
SECTION: Fixation
7. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Biowave settings: 3 x 40 seconds, 150W, Vacuum off, at room temperature.
SECTION: Fixation
8. Post fix cells in 2% Osmium tetroxide in MilliQ water for 30 mins at room temperature, agitating. Tube and well-plate lids must be well-secured during fixation. 

Biowave settings: Repeat cycle 3 times:
2 mins Microwave 100W, Vacuum on, at room temperature
2 mins Microwave off, Vacuum on, at room temperature
SECTION: Fixation
9. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Biowave settings: 3 x 40 seconds, 150W, Vacuum off, at room temperature.
SECTION: Fixation
10. En bloc stain cells with 2% uranyl acetate in MilliQ water overnight at 4 degrees Celsius and then for 1 hour at 50 degrees Celsius, agitating the following day.

Alternatively, en bloc stain cells with 2% uranyl acetate in MilliQ water overnight at 4 degrees Celsius and then Biowave using the following settings:

Biowave settings: Repeat cycle 3 times:
2 mins Microwave 150W, Vacuum on, at 50 degrees Celsius
2 mins Microwave off, Vacuum on, at 50 degrees Celsius

Uranyl acetate enhances contrast of cells 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: Fixation
11. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Biowave settings: 3 x 40 seconds, 150W, Vacuum off, at room temperature. 
SECTION: Fixation
12. Make up a solution of Walton's lead aspartate by dissolving 0.998 g L-aspartic acid into 250 ml MilliQ water and adjust the pH to 3.8 to allow the L-aspartic acid to dissolve more easily. Dissolve 0.066 g lead nitrate in 10 ml L-aspartic acid solution, adjust the pH to 5.5 with potassium hydroxide and stabilise the stain by heating to 60 degrees Celsius for 30 minutes prior to use. L- aspartic acid can be stored for 2 months at 4 degrees Celsius. 

En bloc stain cells with freshly made lead aspartate for 1 hour at 50 degrees Celsius, agitating. 

Biowave settings: Repeat cycle 3 times:
2 mins Microwave 150W, Vacuum on, at 50 degrees Celsius
2 mins Microwave off, Vacuum on, at 50 degrees Celsius

Lead aspartate increases sample conductivity and enhances contrast of tissue by interacting with nucleic acids, proteins and osmium. Unlike lead citrate, en bloc use of lead aspartate does not readily produce electron dense precipitates from exposure to carbon dioxide. Samples contrasted with lead aspartate do not require additional contrasting using lead citrate.
SECTION: Fixation
13. Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating. 

Biowave settings: 3 x 40 seconds, 150W, Vacuum off, at room temperature.
SECTION: Agarose embedding (optional)
15. Gently detach adherent cells from the bottom of the well-plate or petri dish using a cell scraper. 
SECTION: Agarose embedding (optional)
16. 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.
SECTION: Agarose embedding (optional)
17. 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 (optional)
18. Remove the supernatant and add approximately 100 μl (2 drops) of the pre-warmed 4% aqueous agarose to the cell pellet.
SECTION: Agarose embedding (optional)
19. Gently vortex the sample to homogeneously resuspend the cell pellet in pre-warmed 4% aqueous agarose.
SECTION: Agarose embedding (optional)
20. 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 the cells using a benchtop microcentrifuge.
SECTION: Agarose embedding (optional)
21. Solidify the 4% aqueous agarose with cells by cooling at 4 degrees Celsius for 10 minutes.  
SECTION: Agarose embedding (optional)
22. 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 (optional)
23. 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 1 mm3. Carefully place the agarose-embedded cell cubes into a 5 ml tube using forceps.
SECTION: Dehydration and resin infiltration
24. For Steps 24-38, 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. 


Dehydrate in 25% ethanol in MilliQ water for 15 mins at room temperature, agitating.

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

Biowave settings: 40 seconds, 150W, No vacuum, at room temperature.
SECTION: Dehydration and resin infiltration
26. Dehydrate in 75% ethanol in MilliQ water for 15 mins at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum, at room temperature.
SECTION: Dehydration and resin infiltration
27. Dehydrate in 90% ethanol in MilliQ water for 15 mins at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum, at room temperature.
SECTION: Dehydration and resin infiltration
28. Dehydrate in 100% ethanol for 15 mins at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum, at room temperature.
SECTION: Dehydration and resin infiltration
29. Dehydrate in 100% anhydrous ethanol for 15 mins 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, at room temperature.
SECTION: Dehydration and resin infiltration
30. Dehydrate in 100% acetone for 15 mins at room temperature, agitating.

Biowave settings: 40 seconds, 150W, No vacuum, at room temperature.
SECTION: Dehydration and resin infiltration
31. Dehydrate in 100% anhydrous acetone for 15 mins 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, at room temperature.
SECTION: Dehydration and resin infiltration
32. Infiltrate with 25% hard Epon resin in 100% anhydrous acetone overnight at room temperature, on a rotor.

Biowave settings: 3 minutes, 250W, Vacuum on, at room temperature.
SECTION: Dehydration and resin infiltration
33. Infiltrate with 50% hard Epon resin in 100% anhydrous acetone for 6 hours at room temperature, on a rotor.

Biowave settings: 3 minutes, 250W, Vacuum on, at room temperature.
SECTION: Dehydration and resin infiltration
34. Infiltrate with 75% hard Epon resin in 100% anhydrous acetone overnight at room temperature, on a rotor.

Biowave settings: 3 minutes, 250W, Vacuum on, at room temperature.
SECTION: Dehydration and resin infiltration
35. Infiltrate with 100% hard Epon resin for 6 hours at room temperature, on a rotor.

Biowave settings: 3 minutes, 250W, Vacuum on, at room temperature.
SECTION: Dehydration and resin infiltration
36. Infiltrate with 100% hard Epon resin overnight at room temperature, on a rotor.

Biowave settings: 3 minutes, 250W, Vacuum on, at room temperature.
SECTION: Dehydration and resin infiltration
37. Infiltrate with 100% hard Epon resin for 6 hours at room temperature, on a rotor.

Biowave settings: 3 minutes, 250W, Vacuum on, at room temperature.
SECTION: Dehydration and resin infiltration
38. Infiltrate with 100% hard Epon resin overnight at room temperature, on a rotor.

Biowave settings: 3 minutes, 250W, Vacuum on, at room temperature.
SECTION: Dehydration and resin infiltration
39. Transfer samples to labelled plastic embedding moulds, flat bottom BEEM® capsules or standard tip BEEM® capsules containing 100% hard Epon resin. 

For cell monolayers, embed samples in a petri dish by inverting a resin-filled flat bottom BEEM® capsule on top of the cells.
SECTION: Sample mounting and trimming
41. Proceed to Step 43 for mounting and trimming of agarose embedded cells. 
SECTION: Sample mounting and trimming
44. Mount the resin embedded cell monolayer or agarose cell block onto an aluminium SEM specimen pin mount or aluminium rod (10 mm length x 3 mm diameter) using hard Epon resin or fast set liquid epoxy glue. Ensure the cell monolayer is facing up. 
SECTION: Sample mounting and trimming
45. Polymerise the resin overnight in an oven set to 60 degrees Celsius.
SECTION: Sample mounting and trimming
51. For cell monolayers, coat all four sides of the resin block with conductive silver liquid or carbon/graphite paint, taking care not to coat the top imaging surface. Proceed to Step 52. 

For agarose embedded cell blocks, coat all uncut surfaces with conductive silver liquid or carbon/graphite paint, taking care not to coat the top and perpendicular imaging surfaces trimmed with the diamond knife (Steps 48 and 50). Proceed to Step 53.
SECTION: Sample mounting and trimming
53. Sputter coat the entire cell block and specimen pin mount or aluminium rod with 20 nm of gold using a sputter coater. 

For example, Leica EM ACE600 high vacuum sputter coater, equipped with 3 axis motorised stage with planetary drive.
SECTION: Sample mounting and trimming
54. Place the coated cell block or cell monolayer on specimen pin mount or aluminium rod in a focused ion beam scanning electron microscope for preparation of the cell surface for sequential milling and high resolution imaging. 

Example: TFS Helios 5 UX FIB-SEM, equipped with slice and view software.
SECTION: Fixation
14. Proceed to Step 15 for embedding of adherent cells into an agarose block, and to Step 17 for embedding of cells in suspension into an agarose block.

Proceed to Step 24 for the processing of intact cell monolayers. 
SECTION: Sample mounting and trimming
42. Transfer the petri dish containing the embedded cell monolayer and inverted BEEM® capsule into liquid nitrogen briefly using the appropriate PPE, and snap off the BEEM® capsule from the dish.
SECTION: Dehydration and resin infiltration
40. Polymerise the resin in an oven set to 60 degrees Celsius for 48 hours. 
SECTION: Sample mounting and trimming
43. Cut the cell sample out of the resin block using a jewellers saw or razor blade. 

A fume hood must be used when sawing resin to prevent inhalation of fine resin particles. 
SECTION: Sample mounting and trimming
52. Optionally, image the block surface using a stereo microscope mounted with a camera. The image can be used to help locate specific cells within the monolayer using a focused ion beam scanning electron microscope (Step 54). 
SECTION: Sample mounting and trimming
46. For cell monolayers, proceed to Step 51.
SECTION: Sample mounting and trimming
47. Mount the resin-embedded agarose cell block in an ultramicrotome chuck and secure the chuck to a stereo microscope fitted with a chuck mount. Manually trim the top face of the block to approach the cells using a double edged razor blade. 
SECTION: Sample mounting and trimming
48. Transfer the chuck and resin-embedded agarose cell block to a Leica UC7 ultramicrotome and secure in place. Trim the top surface of the block face until a smooth surface through the cell sample has been obtained. Trim manually using an 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 90 degree angle trimming diamond knife. 
SECTION: Sample mounting and trimming
49. To check the region of interest:  
SECTION: Sample mounting and trimming
49.1. 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: Sample mounting and trimming
49.2. 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: Sample mounting and trimming
49.3. Optionally, 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. 

Sections may not require staining because they have been en bloc stained (Steps 10 and 12). 

Toluidine blue stains nucleic acids and proteins, enhancing structural detail of semi thin cell sections.
SECTION: Sample mounting and trimming
49.4. Observe sections with a wide-field light microscope. If you are not within 5  μm of the region of interest, trim deeper and repeat Step 49. 
SECTION: Sample mounting and trimming
50. To create an imaging surface perpendicular to the top surface, trim away the right or left side of the cell block to within 5 um of the region of interest, cutting 100 μm deep. Trim using an 0.5 μm feed and a suitable diamond knife, for example a Diatome 90 degree angle trimming diamond knife. This step can be excluded if you plan to prepare your imaging surface using a FIB-SEM.