Electrode Preparation for sem

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Richter, Hanno -

Electrode Preparation for SEM

  1. Put fresh electrode in 50mM Phosphate Buffer at pH 7.2 with 2% glutaraldehyde and 0.5mM acid.

    1. Use this for conservation of electrode

    2. Can be left overnight

  2. Cut electrode into small 2x2 cm pieces

    1. Perform under Phosphate Buffer without glutaraldehyde

    2. Be sure to wear gloves

  3. Store half of the electrode (2x4 cm) in 50mM Phosphate Buffer at pH 7.2 with 2% glutaraldehyde and 0.5mM acid.

  4. Rinse electrodes for 5 minutes in glutaraldehyde-free Phosphate Buffer

  5. Mix 1% osmium tetroxide in 8mL of 50mM Phosphate buffer with 1 mM Acid for disinfection

    1. Use osmium in the fume hood, it is very reactive with lipid double bonds

  6. Leave it for half an hour.

  7. Test the reactivity of osmium with a small piece of Polyethylene (pipette)

  8. Change buffer one time to 8mL of 50mM Phosphate buffer with 1 mM Acid

    1. Do not use osmium to rinse electrode.

  9. Dehydrate electrode:

  10. Rinse for 5 minutes in Milli-Q to remove salts

  11. Rinse for 5 minutes with increasing concentrations of ethanol

    1. Use ethanol rinses for 5 minutes each at 30%, 50%, 70%, 95%, then 100%

  12. Repeat the 100% ethanol rinse 3 times

  13. The final ethanol wash should be dried with molecular sieves

    1. Molecular sieves activated 8-12 Mesh indirecting Type 3A 2707-01 500g (JT Baker)

    2. Waste osmium goes into vegetable oil.

  14. Put sample into critical point dryer and follow instruction to dry it

  15. Mount dry sample with conductive carbon tape onto stage

  16. Use duco cement to fill the gaps between the glass electrode and the stage

  17. Electrically connect gold layer to the stage using Colloidal graphite with isopropanol

    1. Necessary for sputtering with gold

  18. Sputtering:

    1. Put stage with sample into the sputters

    2. Vacuum should be high to get rid of all solvent vapors from the carbon stage

    3. After good vacuum is reached, flush the chamber with Argon

      1. Use Argon leak valve

      2. Adjust pressure to 0.06 Torr with Argon leak valve

    4. Sputter at ≈ 5mA for 35 min (Voltage =2.2 kV)

  19. SEM:

    1. Hit Vent button → Vacuum

    2. Wait for HT to be ready

    3. press HT – button (high voltage)

    4. Adjust kV with filament

      1. If see peak on screen rising, increase voltage just until peak doesn’t increase anymore

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