Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States
In EM rotary shadowing allows resolution of portions of the specimen that might otherwise have been obscured by a static shadow.
- Make 20µg of protein (per macromolecule of interest to be shadowed) up to a final volume of 500µl with PBS (or 0.155M ammonium acetate, pH7.4) then dialyze overnight against 500ml of 0.155M ammonium acetate (pH7.4)
- Turn on Edward’s 306A coater (power, water, rotary pump, and diffusion pump).
- Prepare the coater for shadowing:
With the main valve closed allow air to enter the bell jar.
- Make sure that the twin electron beam gun power supply is off then wearing sterile gloves detach electrode wires from gun and remove gun from mounting pillar.
- Place gun face up on table, unscrew and remove front shields, then use air gun to clear away loose flaking metal. Remove old tungsten filament and discard. Remove and retain old tungsten rod.
- Reinstall a new acetone-cleaned filament and an acetone-cleaned tungsten rod containing 5cm of 100% platinum (better than platinum-palladium) wrapped about one end. In reinstallation, platinum-wire should be positioned immediately behind filament. Replace front shields.
- Remount gun in chamber at height indicated to give a 9deg angle from gun to stage. Use a metal rod to make sure gun is correctly aimed (angle may be checked with a protractor).
- Use air gun to clean carbon source then reload.
- Check the rotating stage is set at 0deg (graded at back surface of stage).
- Place a piece of filter paper on inside of bell jar to catch evaporated material.
- Replace bell jar; put on implosion protector then evacuate.
- Prepare for spraying on of mica:
Cleave in half previously cut 3mm x 3mm mica pieces and place cleaved surface up inside a 9cm petri dish. Have 6 cleaved pieces/petri dish; keep covered. Optionally cut a cross on cleaved surface with fine forceps.
- Take a microscope slide from ETOH wash and dry. Place 1mm x 1mm pieces of double adhesive tape in line at midpoint of slide, then place slide into another petri dish.
- Place covering paper in fume hood and attach airbrush to air source.
- Add 500µl of glycerol to the 500µl of protein in 0.155M ammonium acetate (pH7.4). Mix gently by moving contents in and out of a plastic pipette, making sure that the glycerol goes into the solution.
- Add mixture to airbrush cup. Before attaching cup to airbrush loosen conical nozzle as much as possible without detachment.
- Attach cup to airbrush, then turn on air supply. place petri dish with cleaved mica in a box in fume hood and remove top of dish exposing mica.
- Aim airbrush at mica standing 2-3 feed away and press trigger to initiate spraying.
- At end of spraying, place top on petri dish; wash out up and airgun with dH20
- Sprayed mica can be stored at 4deg C for one week. For rotary shadowing place two of sprayed mica pieces onto microscope slide containing double adhesive tape.
- Aerate bell jar and attach microscope slide to stage with tape. Replace bell jar and implosion protector; evacuate and turn on film thickness monitor.
- When vacuum reaches 2 x 10-5 mbar turn on rotating stage (120rpm).
- Turn on twin electron gun power supply setting voltage at 2.5kV and setting amperage dial to 4.
- Allow vaccume to return to 2x10-5 mbar then set amperage to 0.
- Turn voltage to 4.0kV and turn amperage up slowly to 50mamp.
If arcing occurs turn amperage down quickly then slowly increase again.
- Once 50mamp is reached open shutter on film thickness monitor and shadow for 30 seconds.
- At end of shadowing close shutter, turn down amperage to 0 and voltage to 0.
Filter paper colour indicates if platinum-palladium evaporated (grey colour). When mamp is over 50 tungsten evaporates giving a light brown colour.
- Put on welders goggles.
- Turn on carbon source and increase to setting 7 watching evaporation. Evaporate for 10 to 15 seconds or almost half of carbon point then turn off. Turn off rotation stage.
- Aerate bell jar and remove glass slide.
- Float replica off of mica by insertion of gH20. Come up from below with copper electron microscope grid. Place grid on filter paper in petri dish and allow to dry.
- Examine in electron microscope.
Klaus Kühn, Hanna Wiedemann, Rupert Timpl, Juha Risteli, Hans Dieringer, Tilman Voss, Robert W. Glanville Macromolecular structure of basement membrane collagens FEBS Letters (1981) 10.1016/0014-5793(81)81012-5