How to prepare QUANTIFOIL® holey carbon film supports

QUANTIFOIL® holey carbon film supports can sometimes have some residual waste material from the production process still attached. If you suspect this waste material may be affecting your data collection, we recommend either washing the grids in organic solvent and/or plasma cleaning prior to use. In addition, glow discharge or plasma treatment will increase the hydrophilicity of the grids. Hydrophilic grids wet more evenly, resulting in a more even layer of ice and better quality grids for data collection. Care should be taken when treating grids with an additional ultrathin carbon layer, as these methods may remove that layer as well as any waste deposits. 

Protocol for washing grids in organic solvent1

We recommend the use of chloroform, ethyl acetate or acetone for washing grids.

  1. Place 4-5 layers of filter paper in a glass Petri dish.
  2. Soak the paper with your choice of organic solvent using a glass Pasteur pipette.
  3. Place the grids, QUANTIFOIL® holey carbon foil side upward, on the saturated filter paper.
  4. Cover the Petri dish and leave the grids in a fume hood overnight.
  5. Inspect the grids and repeat as required.
  6. An additional washing step with deionized water may be desirable to remove any water soluble contaminants.

Glow discharge and plasma cleaning protocols2

Glow discharge and plasma cleaning can not only remove carbonaceous deposits but also makes the surface of grids more hydrophilic, so improving the wetting properties of the grid and therefore their quality.

There are several different protocols for plasma cleaning but in all cases the following precautions should be observed:

  • Prior to the treatment the supports should be inspected under a microscope. All supports should be flat, continuous and without dust or lint. Discard any that do not reach this standard.
  • Plasma treated grids should always be used within 1 hour to prevent contamination accumulating after surface modification.
  • Gloves should be worn when handling anything to be placed in the vacuum chamber of a plasma cleaner to prevent contamination and fingerprints.

Although plasma generated from residual air (glow discharging) can be used, plasmas of controlled composition are recommended, as it they give more reproducible results. We provide protocols utilizing both methods:

Protocol 1: Glow discharging

  1. Place supports in the chamber of a glow discharging apparatus. Supports should be placed in the centre of the stage on a clean glass slide, foil side up. 
  2. Pump chamber to 200 mTorr
  3. Turn on HT to 7 kV, current 28-30 mA
  4. Expose supports for 30 sec.
  5. Turn off the HT and slowly vent the chamber.
  6. Store treated supports in a clean glass Petri dish and use within 1 hour.

Protocol 2: Argon-oxygen plasma treatment

Caution: Poorly-shielded plasmas, or those too high in energy, may damage gold supports by sputtering.

  1. Supports should always be placed in the plasma cleaner in the same location relative to the plasma to maximize reproducibility of exposure between batches. Some facilities have created custom holders to aid with this, but supports can simply be placed directly in the chamber on a clean glass slide, foil side up.
  2. Evacuate chamber to << 0.1 mTorr.
  3. Admit high purity argon and oxygen, in a ratio of 9:1 to a pressure of 21 mTorr
  4. Apply radio frequency plasma with 38 W forward power and ≤ 2 W reverse power. 
  5. Exposure time should be optimized for desired hydrophilicity, but will be 10-60 sec. For most supports, around 20 sec works well.
  6. To prevent damage to very thin films of continuous carbon, use 19:1 argon:oxygen ratio at 35 W forward power for 5-15 sec.
  7. Vent plasma chamber and remove supports, use within 1 hour.

Protocol 3: Amylamine plasma treatment

  1. Place supports on a clean glass slide, foil side up, in the chamber in the center of the stage.
  2. Place a small glass vial containing 0.5 mL of amylamine in the chamber at the edge of the stage.
  3. Pump chamber to below 400 mTorr
  4. Turn on HT and adjust power to form uniform plasma (~90 V, 1.5 A)
  5. Expose supports for 30-60 sec.
  6. Turn off HT and slowly vent the chamber. Discard the vial.
  7. Store supports in a clean glass Petri dish and use within 1 hour.

Protocol 4: Hydrogen plasma treatment

For this protocol, a hydrogen generator should be attached to the input port of the plasma generator.

  1. Use only ultra-high purity hydrogen (>99.999% pure), all stainless steel tubing and fittings (NO plastic), ultra-high purity grade regulators.
  2. Distance of coils to the sample is important, as it affects the energy of the hydrogen species. To avoid damaging a graphene top layer, atoms and ions striking the surface must deliver energy <21 eV. To determine whether chamber this sputter threshold is exceeded, either image test graphene supports at increasing hydrogen doses or use a Langmuir probe to directly measure the energy at the sample.
  3. Prior to the treatment, insert the empty sample holder and evacuate the plasma chamber to << 0.1 mTorr.
  4. Burn the empty holder for 10 min at 100% pure hydrogen (35 W forward power, < 2 W reverse power, pure hydrogen).
  5. Mount the graphene supports in the holder and place into the chamber.
  6. Evacuate the chamber to << 0.1 mTorr.
  7. Treat supports for 5-40 seconds hydrogen plasma, using the same settings as (4).
  8. Remove supports and use immediately.


  1. Grassucci, RA, Taylor, DJ and Frank, J. Nat Protoc 2: 3239-3246 (2007).
  2. Passmore, LA and Russo, CJ. Methods Enzymol. 579: 51-86 (2016).