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

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Making Coverslips by Steve Smith, Cheng- Zhong Zhang and Amy Xu
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    20-Dec-2015
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Transcript of Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

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

Making Coverslips

by Steve Smith, Cheng-Zhong Zhang and Amy Xu

Page 2: 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.

Page 3: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

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.

Page 4: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place coverslips on jig

Page 5: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place jig in laser engraver

Index position to upper left corner

Page 6: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Focus Laser Beam

Position laser lens proper distance from coverslips by raising and lowering the table.

Page 7: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Close lid and engrave!

Set printer for 5 copies for deeper engraving

Page 8: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Poke out holes

Use sharp probe and metal backup plate

Page 9: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Making a Gasket

by Steve Smith, Cheng-Zhong Zhang and Amy Xu

Page 10: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Nescofilm with empty roller

Page 11: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Thread Nescofilm through rollers

Suspend Nescofilm flat and taut, far away from any surface (including paper backing)!

Page 12: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place roller in Laser Engraver

Page 13: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Focus Laser Beam

Position laser lens proper distance from Nescofilm surface by raising and lowering the table.

Page 14: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Close lid and engrave!

Smoke is sucked up the vent if your fan is working properly

Page 15: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Remove stray Nescofilm

Page 16: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Advance Nescofilm roller

Page 17: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Pulling a Pipette

by Steve Smith, Cheng-Zhong Zhang and Amy Xu

Page 18: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Clamp Glass Tubing

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

Page 19: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Centering the tube

Shift the filament to center the glass tube.

Page 20: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

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.

Page 21: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Remove pipette

Do not touch the tip to anything!

Page 22: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Inspect pipette tip

Carry pipette on a glass slide and examine on a dissecting microscope.

Page 23: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Pipette tip

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

Page 24: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Assembling a Microchamber

by Steve Smith, Cheng-Zhong Zhang and Amy Xu

Page 25: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Trim the gaskets

Use sharp, clean scissors to cut two gaskets.

Page 26: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Lay gasket on coverslip with no holes

Page 27: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Lay gasket on coverslip with 6 holes

Align holes

Page 28: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Flatten gaskets

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

Page 29: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place pipette in middle

Transfer pipette tip on a 1mm thick glass slide.

Page 30: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Pipette tip placement

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

Page 31: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Cut dispenser tubes

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

Page 32: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place dispenser tubes

“Downstream” is to the left of the pipette.

Page 33: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

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.

Page 34: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Make a sandwich

Align gaskets carefully without moving tubes! Good luck

Page 35: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Press ends together

Gently

Page 36: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place glass slide on top of sandwich

Use a clean 1mm glass slide

Page 37: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Press on glass slide to flatten sandwich

With considerable force

Page 38: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Pick up sandwich and slide together

Don’t break the pipette!

Page 39: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place on heated plate

Cover the hotplate with clean aluminum foil. T = 120o C

Page 40: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

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.

Page 41: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Place microchamber on frame

Align chamber with holes facing down

Page 42: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Tighten microchamber on frame

Press down in the middle of the clamp while gently tightening the screws.

Page 43: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Check alignment of chamber and frame holes

Adjust clamps as necessary

Page 44: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Thread PE10 tubing onto pipette

Tighten bottom clamp with pipette slot facing down.

Page 45: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Dispense small amount of epoxy

Norland UV cure optical adhesive

Page 46: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Touch epoxy to end of tubing

Page 47: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Epoxy enters tube by capillary action

Be careful epoxy does not run too far and clog back end of pipette!

Page 48: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

UV crosslink epoxy

2-5 minutes in crosslinking chamber oven

Page 49: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Screw in nylon tube fittings

Rinse out fittings first

Page 50: Making Coverslips by Steve Smith, Cheng-Zhong Zhang and Amy Xu.

Completed microchamber

Congratulations!