MATERIALS

1) Round (preferred) or square glass cover slip: 12 mm in diameter; order from Ted Pella cat# 26023, 0.25oz, $13.40

2) Stop: to hold or support cover slip and carbon conductive tab; obtain from staff

3) Conductive carbon tabs: 12 mm in outer diameter; to adhere cover slip to stop; order from Ted Pella cat#16084-1, (1/4 oz)

4) Poly-L-Lysine: to adhere organic material to cover slip; order from Sigma cat# P8920, 0.1% wt/vol aqueous solution with preservative added, store at room temperature, $40.00

5) 0.1M Sodium Phosphate Buffer A:
- Dissolve 27.6 g sodium phosphate monobasic NaH2PO4*H2O in 500 mL ddH2O
- pH to 7.2 with 10N NaOH (around 14 mL needed)
- Bring to final volume of 1000 mL with ddH2O

6) 0.2M Sodium Phosphate Buffer B:
- Dissolve 53.6 g sodium dibasic NaHPO4*7H2O in 500 mL ddH2O
- pH to 7.2 with 10N NaOH (around 14 mL needed)
- Bring to final volume of 1000 mL with ddH2O

7) Solution A: 3% Glutaraldehyde:
- 6 mL of 50% glutaraldehyde
- 94 mL 0.1M sodium phosphate buffer A, pH 7.2 (final volume of 100 mL)

8) Solution B: 1% Osmium Tetroxide (see protocol for prep of OsO4 stock solution below)*:
- *make and use in hood; a 1:1 volume solution (500 uL of each solution)
- 2% osmium tertoxide stock (2 g in 100 mL ddH2O)
- 0.2M sodium phosphate buffer B, pH 7.2
- label excess for disposal by EHS

Protocol for Cell Fixation:
Scanning Electron Microscopy

1) Collect 1 mL of cells at 1.0 OD in a 1.5 mL microfuge tube; add 60 uL of 50% Glutaraldehyde to cells and let sit for 1 hour. Or use 300 uL of Solution A
2) Pellet by centrifuging at 10,000 rpm for 5 min; resuspend pellet with 1 mL Solution A; repeat step #2 three more times.
3) To sterilize cover slip, soak with 95% ethanol, then flame (work with cover slip on a kim wipe using tweezers).
4) Gold/Palladium plate the cover slip (*see protocol for Au/Pd plating; remember which side is plated; do not scratch surface).
5) Post fix cells: remove solution A; resuspend pellet with 500 uL of solution B and let sit for 20 min; cell pellet will become black in color; spin cells at 10,000 rpm for 5 min- remove supernatant immediately as cells will resuspend themselves (supernatant is toxic so discard properly).
6) Resuspend cell pellet in 1 mL ddH2O; spin for 5 min at 10,000 rpm; remove supernatant; repeat step #6 three more times.
7) Spot 10 uL of poly-L-lysine onto cover slip and allow to air dry (make sure to use the Gold/Palladium plated side).
8) Spot 10 uL of washed cells onto poly-L-lysine treated side; wait 3 min for cells to settle; cells should bind to poly-L-lysine.
9) Dehydrate cells (fixed on cover slip) with 30%, 50%, 70%, 95%, and 100% ethanol washings (ex: 30% ethanol is 6 mL 100% ethanol with 14 mL ddH2O). Place cell-fixed cover slip in slide rig using tweezers. (don’t scratch cover slip surface!) Fill 50 mL falcon tubes filled with 20 mL of the appropriate % ethanol (use more ethanol if samples aren’t completely covered). Put slide rig with cover slip inside falcon tubes (starting with 30% ethanol washing). Let rig sit in washing solution for 5 min; repeat 1-4 times (make new washing solutions for each repeat).
10) Leave samples in the slide rig in 100% ethanol prior to critical point drying.

Protocol for Critical Point Drying
Scanning Electron Microscopy

1) Place a small amount of solvent i.e. 100% ethanol (acetone if samples are in acetone) in the pressure chamber, and quickly transfer the specimen holder into the chamber, and screw on the pressure chamber cap. Don’t allow the ethanol to evaporate it may cause the sample to dry.
2) Only 4 round coverslip samples per use. Stack 1 slip (cells facing up) per wire mesh basket and continue to add ethanol as necessary so that level is reached just below the inner portion of the chamber cap after final slip and basket are added.
3) Cool the chamber with cold water by filling the metal beaker ½ full of ice and add water so that level is just below the bottom edge of the pressure chamber cap. Insert thermometer.
4) With the Inlet and Outlet valves closed, open the CO2 tank valve approximately 1 full turn.
5) Slowly open the Inlet valve, the pressure of the chamber will rise to about 850 psi. Then open the inlet valve about two full turns. Wait approximately 10 min for equilibration.
6) Slowly open the Outlet valve to exhaust the mixture of CO2 and ethanol.
7) After 2-3 minutes, close the Outlet valve and then the Inlet valve.
8) Turn on the hot tap water in sink.
9) After 5 minutes, slowly open the inlet valve for 2 full turns and then the Outlet valve (notice the deposit of ice on the the tubing). This will flush the system. “CO2 Snow is seen after 5 minutes or so”.
10) Close the Outlet valve, and then the Inlet valve and CO2 tank valve.
11) Dump the ice water
12) Place the beaker under the hot water for several minutes. Also place a thermometer in the beaker to monitor the water temp. Fill the beaker with hot water (50-60°C ). Leave the thermometer in the beaker.
13) Place the beaker with the hot water on to the critical point apparatus and immerse the pressure chamber. Max temperature obtainable is about 55°C. Pressure will rise normally to 1500-2000 psi. (If pressure goes above 1800-2000 psi, open the outlet valve very slowly to reduce pressure and then close the Outlet valve). Careful, need to observe this entire step due to high pressure. At the critical point when equilibrium is reached, there are no surface tensions between the liquid and the gas phases of the CO2.
14) After 5-10 minutes, open the Outlet valve slowly to reduce pressure in the chamber to atmospheric over a period of about 5 minutes.
15) While waiting, place a carbon conductive tab on to the stop. This will hold the round glass coverslip sample once it is dry and removed from the drying chamber.
16) When pressure is zero, remove the beaker and dry off the pressure chamber. Use the edge of a paper towel to absorb water from the thin region between the bottom edge of the pressure chamber cap and the chamber itself. Unscrew the cap and remove specimen holder (i.e. wire mesh holder with sample inside using tweezers). Try to grab the basket with tweezers not the glass sample.
17) The samples can never become wet again even after plating.
18) Now place the sample (glass cover slip w/ cells face up) onto the carbon conductive tab.
19) Repeat the Gold/Palladium plating of the cover slip (*see protocol for Au/Pd plating; remember which side is plated; do not scratch surface).

Protocol for Gold/Palladium (Gold/Pd) plating
Scanning Electron Microscopy

1) Place samples on the metal plate under the bell jar
2) Place the bell evenly on to the plating apparatus
3) Turn the vacuum knob all the way to the right
4) Turn the “decrease knob” all the way to the right
5) Turn the power on
6) Rotate the time (minutes knob) to 8 or 9 minutes
7) Turn the high voltage control on
8) Increase the “decrease knob” until 10 milliamps is displayed in the milliamps readout. *note this knob has to be continuously rotated during high voltage on.
9) Plate while at 10 milliamps for 2 minutes (longer if charging is noticed). Vaccuum is approximately 250 millitorr.
10) After 2 minutes is complete then rotate the time (minutes knob) to 0
11) Turn the high voltage control off
12) Turn the power off
13) Rotate the vacuum knob all the way to the left until all vacuum pressure is released
14) Remove the bell jar and collect samples
15) Samples are now ready for viewing with the SEM

Protocol for SEM machine usage
Scanning Electron Microscopy

1) Turn SEM machine on (usually on all the time)
2) Turn the SEM computer on if not on yet
3) Release the vacuum pressure (press green EVAC button on front of SEM machine)
4) Wait for vacuum pressure indicator on computer to turn completely red (from blue to green to red) (*verify color)
5) Rotate the sample height adjust knob so that the two squares on the working distance dial are aligned
6) Open the sem chamber (SLOWLY) so that if the sample is too high or low it will not hit an important piece inside the chamber
7) Next insert sample(s) up to 3 onto the sample holder and tighten into place with the provided hex wrench.
8) Verify the height of the sample holder by comparing with the height of the standard (a metal cylinder bar/rod) usually next to the SEM on the computer desk. The height of the holder should be equal to the height of the metal bar.
9) Place a small piece of conductive carbon tab tape on the 3 sample holder. Use an arrow shape. This will help indicate the direction of rotation i.e. helps identify the sample you are looking at. The sample cover slip can also be marked carefully with a sharp object.
10) Insert the sample holder into the SEM and slide the chamber closed
11) Press the EVAC button again this time the colors go from red to green to blue. Wait for the color indicator bar on the computer screen to turn completely blue.
12) Click the mouse on HV then click AFS and Auto then choose 20kX
13) Set WD to 7.5
14) Vacc = 25.0 kV
15) Align with stigmator
16) Set the contrast. Kit Lee – “Contrast is key”. A good mid range is required.
17) Focus the sample at minimum magnification (60 to 200x) to see direction of conductive carbon tape and thus can select sample to look at.
18) View sample by either mouse clicks on computer or the hand operated controller and dials on the SEM
19) Adjust horizontal and vertical positioning with dials (corresponding H and V arrows) on SEM
20) Adjust magnification and focus with corresponding knobs
21) Adjust contrast and brightness as req.
22) Select scan speed for desired level of clarity (mouse only on screen) 2 speeds per level icon
23) Set working distance knob (located on SEM to 6.1 mm for 15 to 20kX viewing)
24) Use the rotating knob (located on the SEM) to choose from the 3 samples (rotates the sample holder)
25) Focus on something interesting and magnify. After full magnification adjust the stigmator until the sharpest i.e. clearest image is achieved. Then view again at slow scan speed it should be a really good image if contrast, focus, stigamtor, working distance, and voltage are each set properly.
26) If a picture is desired click SCAN on computer screen (verify) and save the picture in the appropriate directory. It will be necessary to aquire the saved images later using a 100mb disk from a different computer in the microscopy lab. It may also be possible to retrieve the pictures from the network “Bdl-net 1\UserVol1’(J:)”
27) When finished viewing samples click HV off, then EVAC then wait till ok to open chamber
28) Align squares on WD knob and pull chamber open slowly
29) Remove samples
30) Close chamber and press EVAC again to restore vacuum (verify)

Protocol for Solid Environmental Sample Preparation
Scanning Electron Microscopy

1) Collect particles of sample the more fine grain the better
2) Less is more
3) Attach a carbon conductive tab to the stop
4) Attach the particles directly to the other sticky side of the carbon conductive tab
5) Plate the side with the sample particles using the protocol for Gold/Pd plating
6) View with SEM using the procedure for SEM viewing

Protocol for 2% Osmium Tetroxide OsO4 Preparation
Scanning Electron Microscopy

MATERIALS:

1) 1g OsO4 in glass vial: order from Sigma
2) 50 mL d2H2O: in glass bottle

PROCEDURE (Osmium tetroxide (OsO4) Preparation) In Hood:

1) Run hot tap water over glass vial for several minutes until the OsO4 crystals are melted
2) Score around the middle of the vial with a small glass cutter
3) Snap the vial into two halves
4) Drop both halves of the vial into the bottle with the d2H2O
5) Run the bottle under hot tap water for several minutes to liquefy the molten glob of OsO4
6) Place the bottle into an ultrasound bath for 5 to 10 minutes to vibrate the OsO4 into solution
7) This forms a 2% OsO4 stock solution

Protocol for On-Coverslip Sample Preparation
Scanning Electron Microscopy

1) Use a petri-dish in hood
2) Gold/Pd plate a coverslip using the protocol above
3) Spot 10uL poly-L-Lysine onto coverslip and allow to dry
4) Spot 10uL of cells from culture (at 1.0 o.d.) let stand 3 minutes to attach to poly-L-Lysine (don’t allow to dry)
5) Spot 10uL of Solution A on to coverslip (don’t allow to dry)
6) Spot 10uL of the 1% OsO4 solution (don’t allow to dry)
7) Perform the ethanol washes (don’t allow to dry)
8) Perform the critical point drying
9) Attach a carbon conductive tab to the stop
10) Attach sample to the tab on the stop
11) Gold/Pd plate the sample
12) View under SEM