Chapter 2: The FSU SEM User Manual

How to use the SEM

The SEM in the FSU Biology department is a Cambridge Instruments Stereoscan S100 Scanning Electron Microscope that does all of its imaging using secondary electrons. The room housing the electron microscope and related equipment is housed in the first door on the right off the stairwell which passes by the Greenhouse in Tawes hall. The first step that needs to be taken before using the SEM is to prepare the specimen for viewing in the SEM. There are three conditions that a sample must meet in order to be viewed in the SEM and to prevent damage to the microscope:

1.  The sample should be desiccated (i.e. contain no water) and should be free of solvents, oil from human skin, and anything else that could boil away in a vacuum.
2. The sample MUST be electrically conductive.
3. The sample must be properly mounted on a specimen stub.
   

The first two criteria are generally more of a problem for someone who wishes to view biological samples and for someone who wishes to view a non-conductive non-biological material (ceramics or plastics, for example ).

The procedure for preparing a biological sample, such as some sort of plant material is as follows:⁴⁸

1. Cut the plant up into manageable pieces.
2. Put the pieces into a vial with a cap.
3. Pipette Gluteraldehyde fixative solution into vial until half full (helps to preserve cellular structures).
4. Let sit for 2 hours.
5. Pipette out Gluteraldehyde.
6. Add a 35% Ethanol solution to dehydrate sample.
7. Let sit for 1 hour.
8. Pipette out the 35% Ethanol solution and replace with a 70% Ethanol solution.
9. Let sit for one hour.

The specimen should then be allowed to dry so that all the Ethanol can evaporate from the sample. The sample now should be treated in a sputter coater to make it electrically conductive. Click here to see what a sputter coater looks like.

What is a Sputter Coater?

Any sample to be viewed using the SEM must be electrically conductive.

If the experimenter wishes to view a metallic or semiconducting sample, the sample is just affixed to the viewing stage with conductive carbon cement. However, if the researcher wants to view anything non-conductive under the SEM(biological samples, ceramics, some plastics, etc.), then the sample must be coated with an extremely thin, evenly applied conducting film of gold. This is basically what the sputter coater does. The sputter coater coats a sample by depositing a thin film of ionized gold atoms on the specimen in an Argon atmosphere(to prevent the gold ions from reacting with atmospheric gases, since Argon is a noble gas and thus inert).

Proper Usage of the Sputter Coater:

1. Lift the lid of the sputter coater.
2. Place prepared specimen to be coated on table inside the sputter coater.
3. Replace the lid.
4. The black switch is the Leak Valve. Flip it down (i.e. towards the operator).
5. Turn on the pump switch.
6. Turn on the pressure gauge switch.
7. Turn on the Argon tank via the screw knob on the top of the tank.
8. Screw in the silver "Scientific Company" brass handle on the sputter coater.
9. Leak Argon into the chamber by turning the small silver Knob on the sputter coater. Let the Argon leak into the chamber for a few minutes, until the pressure gauge reads 250.
10. Open the brass screw on the clear plastic hose by screwing it counter-clockwise until you hear the gas begin to leak into the chamber. This is replacing the air in the sputter coater with Argon.
11. Close the small silver leak knob on the sputter coater
12. Turn on the heater switch on top of the sputter coater.
13. Turn the black knob on the front panel of the sputter coater 1/2 way, until a purple glow is visible. Look for an eveness of purple color and DO NOT let it arc. This will result in ionized argon, which is chemically reactive and the result will be a sample which is not evenly coated with gold.
14. Set the timer for 2 minutes.
15. After the 2 minutes have elapsed, turn down the black knob.
16. Switch off the output gauge.
17. Turn off the argon
18. Switch off the pump.
19. Open the black leak knob slowly.
20. Remove samples from the sputter coater.

For most physics and engineering students, however, sample preparation is actually quite easy. Most specimens that would be of interest to students in either of these majors are already conductive, such as metals and semiconductors. The procedure for preparing electrically conductive samples is as follows:

1. Put on rubber gloves and wash hands to remove powder from the outer surface of the gloves.
2. Clean specimen stubs under the ventilation hood in the SEM lab with a lens cloth saturated with acetone and allow the stubs to dry for approximately 10 to 15 minutes.
3. Clean sample with acetone if possible. Some substances react negatively with acetone.
4. If the sample can not be cleaned with acetone, use another cleaning method (i.e. a different solvent, polishing with special abrasives, or ultrasonic cleaning).
5. Mix the conductive carbon cement well under the ventilation hood.
6. Affix the sample(s) to the stub(s) using the cement. Use plenty of cement (the electron beam has kinetic energy!), but do not get any cement on an area of interest.
7. Allow the cement to dry for 15 to 20 minutes.
   
   NOTE: It is an excellent idea to make a diagram of the box holding your stubs so you know which sample is which!
   
   
8. Place in the SEM (while wearing rubber gloves) using the stub tweezers located on the SEM. The stubs go in a stage similar to the one in the following picture (the stub holder on the stage is the copper colored area) :

After the samples have been properly prepared, you are now ready to use the SEM. There are a few important procedures to follow to insure that good imaging results are obtained and the SEM is not damaged. Some of these advisories will not become clear until you read the next two sections, but I will mention them now so they will be fresh in your mind.

• The SEM is ALWAYS kept pumped down (evacuated) when not in use. This helps to prolong the life-span of the filament and o-rings. Therefore, NEVER EVER just open the sample chamber door! The sudden influx of air will almost certainly damage the SEM.
  
• NEVER rapidly crank up the filament to its maximum voltage. This will cause the filament to burn out.
  
• ALWAYS wear rubber gloves when putting anything into or taking anything out of the SEM.
  
• NEVER vent the sample chamber when the filament is on. This will burn out the filament.
  
• ALWAYS turn the filament off before disengaging the beam. This also helps prolong the life of the filament.
  
• If you have a question about operating the SEM, sample preparation, or need assistance OF ANY KIND call Dr. Morton (his number is posted above the phone in the sample preparation lab): 687-4355; or Dr. Hoffman: 687-4160 and ASK!

Following the above caveats will help to ensure a successful session with the SEM for yourself and others. The two sections showing the procedures for verifying that the SEM is functional and for imaging with the SEM are located at the end of this report in flow chart format. These procedures are also included in the instructional manual for using the SEM that is located in the same room as the SEM.

To:
Table of Contents
Chapter 1: How the SEM Works
Imaging with the SEM