TITLE: Preparation of Tissue 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 tissue 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.

Dissect out the tissue of interest on a Teflon plate or dental wax sheet using fine forceps and a scalpel blade at room temperature, and keeping the tissue submerged in Karnovsky's fixative, 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4). Tissue pieces should be no larger than 1 mm cubed to ensure sufficient infiltration of fixative, solvents and resin.  
SECTION: Fixation
2. Place the tissue pieces in 5 ml tubes, 15 ml Falcon® tubes or a well-plate containing 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer and fix overnight at 4 degrees Celsius or for 2-4 hours at room temperature, on a rotor. 
SECTION: Fixation
3. Wash with 0.1M cacodylate buffer, 3 x 10 minutes at room temperature, agitating. 
SECTION: Fixation
4. 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 3) prior to post-fixation (Step 4) to avoid this. 

The following Steps 4-29 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 tissue in 2% osmium tetroxide in 0.1M cacodylate buffer for 1.5 hours at room temperature, agitating. Tube and well-plate lids must be well-secured during fixation. 

The microwave regime described below (Steps 4, 5, 7, 9, 11, 13) 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
5. Post fix the tissue in 1.5% potassium ferrocyanide in 0.1M cacodylate buffer for 1.5 hours 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 artefactual precipitates. Potassium ferricyanide can be used as an alternative to potassium ferrocyanide and remains a clear, amber colour if mixed with osmium. 
SECTION: Fixation
6. 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
7. Incubate the tissue in 1% Thiocarbohydrazide in MilliQ water for 45 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 9, thereby enhancing contrast of lipid membranes. The increase in osmium content also makes tissue more conductive.
SECTION: Fixation
8. 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
9. Post fix tissue in 2% osmium tetroxide in MilliQ water for 1.5 hours at room temperature, agitating. Tube or 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
10. 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
11. En bloc stain tissue with 2% uranyl acetate in MilliQ water overnight at 4 degrees Celsius and then for 2 hours at 50 degrees Celsius, agitating the following day.

Alternatively, en bloc stain tissue 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 tissue 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
12. 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
13. 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 tissue with freshly made lead aspartate for 2 hours 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
14. 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: Dehydration and resin infiltration
15. For Steps 15-29, 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
16. 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
17. 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
18. 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
19. 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
20. 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
21. 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
22. 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
23. 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
24. 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
25. 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
26. 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
27. 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
28. 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
29. 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
30. Transfer samples to labelled plastic embedding moulds, flat bottom BEEM® capsules or standard tip BEEM® capsules containing 100% hard Epon resin.
SECTION: Sample mounting and trimming
32. Cut the tissue 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
33. Mount the resin embedded tissue 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 region of interest is facing up and the sample is mounted in the correct orientation for imaging.
SECTION: Sample mounting and trimming
34. Polymerise the resin overnight in an oven set to 60 degrees Celsius.
SECTION: Sample mounting and trimming
40. Sputter coat the entire tissue 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
35. 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 top face of the block to approach the region of interest using a double edged razor blade. 
SECTION: Sample mounting and trimming
39. Coat all uncut surfaces of the tissue block 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 36 and 38).
SECTION: Sample mounting and trimming
36. Transfer the chuck and resin-embedded tissue block to a Leica UC7 ultramicrotome and secure in place. Trim the top surface of the block face until a smooth surface no more than 5  μm from the region of interest 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
37. To check the region of interest:  
SECTION: Sample mounting and trimming
37.1. 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
37.2. 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
37.3. 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 11 and 13). 

Toluidine blue stains nucleic acids and proteins, enhancing structural detail of semi thin tissue sections.
SECTION: Sample mounting and trimming
37.4. 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 37. 
SECTION: Sample mounting and trimming
38. To create an imaging surface perpendicular to the top surface, trim away the right or left side of the tissue 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. 
SECTION: Sample mounting and trimming
41. Place the coated tissue block on specimen pin mount or aluminium rod in a focused ion beam scanning electron microscope for sequential milling and high resolution imaging. 

Example: TFS Helios 5 UX FIB-SEM, equipped with slice and view software.
SECTION: Dehydration and resin infiltration
31. Polymerise the resin in an oven set to 60 degrees Celsius for 48 hours.