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Making Coverslips

Making Coverslips. by Steve Smith, Cheng- Zhong Zhang and Amy Xu. Inspect coverslip for flatness. Interference fringes most visible in fluorescent light through a magnifying glass. Interference fringes.

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Making Coverslips

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  1. Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu

  2. Inspect coverslip for flatness Interference fringes most visible in fluorescent light through a magnifying glass.

  3. Interference fringes Each fringe reflects a ½ λ change in thickness. Few stripes are best- this coverslip is OK. Beware of coverslips with so many fringes they appear invisible.

  4. Place coverslips on jig

  5. Place jig in laser engraver Index position to upper left corner

  6. Focus Laser Beam Position laser lens proper distance from coverslips by raising and lowering the table.

  7. Close lid and engrave! Set printer for 5 copies for deeper engraving

  8. Poke out holes Use sharp probe and metal backup plate

  9. Making a Gasket by Steve Smith, Cheng-Zhong Zhang and Amy Xu

  10. Nescofilm with empty roller

  11. Thread Nescofilm through rollers Suspend Nescofilm flat and taut, far away from any surface (including paper backing)!

  12. Place roller in Laser Engraver

  13. Focus Laser Beam Position laser lens proper distance from Nescofilm surface by raising and lowering the table.

  14. Close lid and engrave! Smoke is sucked up the vent if your fan is working properly

  15. Remove stray Nescofilm

  16. Advance Nescofilm roller

  17. Pulling a Pipette by Steve Smith, Cheng-Zhong Zhang and Amy Xu

  18. Clamp Glass Tubing Carefully clamp pipette at top and bottom, threading through the filament. Don’t touch glass tubing in the center. Fingerprints = bad.

  19. Centering the tube Shift the filament to center the glass tube.

  20. Shield from air currents and PULL! Cover pipette puller with a plastic beaker and turn on power supply to heat the filament. Weight should drop in ~10 seconds. Faster heating = smaller tips.

  21. Remove pipette Do not touch the tip to anything!

  22. Inspect pipette tip Carry pipette on a glass slide and examine on a dissecting microscope.

  23. Pipette tip Tip should be too small to resolve in a dissecting microscope.

  24. Assembling a Microchamber by Steve Smith, Cheng-Zhong Zhang and Amy Xu

  25. Trim the gaskets Use sharp, clean scissors to cut two gaskets.

  26. Lay gasket on coverslipwith no holes

  27. Lay gasket on coverslip with 6 holes Align holes

  28. Flatten gaskets Press gaskets against glass to make them stick. Do not get fingerprints in the center.

  29. Place pipette in middle Transfer pipette tip on a 1mm thick glass slide.

  30. Pipette tip placement Place glass tube over alignment holes. Place tip halfway through center channel. Press glass tube into gasket to make it stick.

  31. Cut dispenser tubes Cut ~2.5 mm pieces from long tube. Secure on double sided tape.

  32. Place dispenser tubes “Downstream” is to the left of the pipette.

  33. Dispenser tube placement 2.5 mm long dispenser tubes placed at a diagonal to adjust length into channel. Too close to the walls = fills with melted Nescofilm. Too long = catches bubbles.

  34. Make a sandwich Align gaskets carefully without moving tubes! Good luck 

  35. Press ends together Gently

  36. Place glass slide on top of sandwich Use a clean 1mm glass slide

  37. Press on glass slide to flatten sandwich With considerable force

  38. Pick up sandwich and slide together Don’t break the pipette!

  39. Place on heated plate Cover the hotplate with clean aluminum foil. T = 120o C

  40. Place heated weight on glass slide From bottom to top: hotplate, aluminum foil, microchamber, 1mm glass slide, aluminum foil, heated aluminum block (~ 250 grams), aluminum foil handles. Heat for 5 minutes.

  41. Place microchamber on frame Align chamber with holes facing down

  42. Tighten microchamber on frame Press down in the middle of the clamp while gently tightening the screws.

  43. Check alignment of chamber and frame holes Adjust clamps as necessary

  44. Thread PE10 tubing onto pipette Tighten bottom clamp with pipette slot facing down.

  45. Dispense small amount of epoxy Norland UV cure optical adhesive

  46. Touch epoxy to end of tubing

  47. Epoxy enters tube by capillary action Be careful epoxy does not run too far and clog back end of pipette!

  48. UV crosslink epoxy 2-5 minutes in crosslinking chamber oven

  49. Screw in nylon tube fittings Rinse out fittings first

  50. Completed microchamber Congratulations!

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