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Electrode Preparation for sem
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tarix | 02.03.2018 | ölçüsü | 5,97 Kb. | | #29555 |
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Electrode Preparation for SEM
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Put fresh electrode in 50mM Phosphate Buffer at pH 7.2 with 2% glutaraldehyde and 0.5mM acid.
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Use this for conservation of electrode
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Can be left overnight
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Cut electrode into small 2x2 cm pieces
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Perform under Phosphate Buffer without glutaraldehyde
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Be sure to wear gloves
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Store half of the electrode (2x4 cm) in 50mM Phosphate Buffer at pH 7.2 with 2% glutaraldehyde and 0.5mM acid.
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Rinse electrodes for 5 minutes in glutaraldehyde-free Phosphate Buffer
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Mix 1% osmium tetroxide in 8mL of 50mM Phosphate buffer with 1 mM Acid for disinfection
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Use osmium in the fume hood, it is very reactive with lipid double bonds
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Leave it for half an hour.
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Test the reactivity of osmium with a small piece of Polyethylene (pipette)
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Change buffer one time to 8mL of 50mM Phosphate buffer with 1 mM Acid
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Do not use osmium to rinse electrode.
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Dehydrate electrode:
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Rinse for 5 minutes in Milli-Q to remove salts
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Rinse for 5 minutes with increasing concentrations of ethanol
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Use ethanol rinses for 5 minutes each at 30%, 50%, 70%, 95%, then 100%
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Repeat the 100% ethanol rinse 3 times
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The final ethanol wash should be dried with molecular sieves
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Molecular sieves activated 8-12 Mesh indirecting Type 3A 2707-01 500g (JT Baker)
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Waste osmium goes into vegetable oil.
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Put sample into critical point dryer and follow instruction to dry it
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Mount dry sample with conductive carbon tape onto stage
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Use duco cement to fill the gaps between the glass electrode and the stage
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Electrically connect gold layer to the stage using Colloidal graphite with isopropanol
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Necessary for sputtering with gold
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Sputtering:
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Put stage with sample into the sputters
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Vacuum should be high to get rid of all solvent vapors from the carbon stage
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After good vacuum is reached, flush the chamber with Argon
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Use Argon leak valve
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Adjust pressure to 0.06 Torr with Argon leak valve
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Sputter at ≈ 5mA for 35 min (Voltage =2.2 kV)
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SEM:
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Hit Vent button → Vacuum
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Wait for HT to be ready
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press HT – button (high voltage)
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Adjust kV with filament
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If see peak on screen rising, increase voltage just until peak doesn’t increase anymore
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