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

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.

Place coverslips on jig

Place jig in laser engraver

Index position to upper left corner

Focus Laser Beam

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

Close lid and engrave!

Set printer for 5 copies for deeper engraving

Poke out holes

Use sharp probe and metal backup plate

Making a Gasket

by Steve Smith, Cheng-Zhong Zhang and Amy Xu

Nescofilm with empty roller

Thread Nescofilm through rollers

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

Place roller in Laser Engraver

Focus Laser Beam

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

Close lid and engrave!

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

Remove stray Nescofilm

Advance Nescofilm roller

Pulling a Pipette

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.

Centering the tube

Shift the filament to center the glass tube.

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.

Remove pipette

Do not touch the tip to anything!

Inspect pipette tip

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

Pipette tip

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

Assembling a Microchamber

by Steve Smith, Cheng-Zhong Zhang and Amy Xu

Trim the gaskets

Use sharp, clean scissors to cut two gaskets.

Lay gasket on coverslip with no holes

Lay gasket on coverslip with 6 holes

Align holes

Flatten gaskets

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

Place pipette in middle

Transfer pipette tip on a 1mm thick glass slide.

Pipette tip placement

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

Cut dispenser tubes

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

Place dispenser tubes

“Downstream” is to the left of the pipette.

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.

Make a sandwich

Align gaskets carefully without moving tubes! Good luck

Press ends together

Gently

Place glass slide on top of sandwich

Use a clean 1mm glass slide

Press on glass slide to flatten sandwich

With considerable force

Pick up sandwich and slide together

Don’t break the pipette!

Place on heated plate

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

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.

Place microchamber on frame

Align chamber with holes facing down

Tighten microchamber on frame

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

Check alignment of chamber and frame holes

Adjust clamps as necessary

Thread PE10 tubing onto pipette

Tighten bottom clamp with pipette slot facing down.

Dispense small amount of epoxy

Norland UV cure optical adhesive

Touch epoxy to end of tubing

Epoxy enters tube by capillary action

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

UV crosslink epoxy

2-5 minutes in crosslinking chamber oven

Screw in nylon tube fittings

Rinse out fittings first

Completed microchamber

Congratulations!