SEM METHODS

 

 

 PROTOCOLS FOR SEM TISSUE PREPARATION

 

 

The methods outlined below for tissue fixation are very general and should not be used in place of methods published for the tissue you are examining. For people experienced in TEM tissue preparation, the initial stages of fixation and dehydration to absolute ethanol are identical except that larger pieces of tissue may be used.

 

CAUTION: Glutaraldehyde and osmium tetroxide are possible carcinogens. Gloves must be worn when handling these chemicals. The chemicals must be used in designated areas of the hood in SCST147 and disposed of in the proper containers. Pasteur pipets that contact these chemicals should also be disposed of in the labeled container in the hood.

 

Fixation:

 

1. Tissue should be immersed in 3% buffered glutaraldehyde for 2-3 hours (fixation times of up to 24 hours may be necessary for some tissues). Make sure that the appropriate label accompanies each vial of tissue you prepare.

 

2. Wash 2-3 times in buffer (10-15 mins each change) to remove all traces of glutaraldehyde.

 

3. Post-fix in 2% osmium tetroxide in buffer for 1-2 hours for animal tissue and overnight (12-18 hours) for plant tissue. Post fixation may be omitted in some protocols for SEM.

 

4. Dehydrate tissue through graded ethanols:

 

            50%..............15-30 mins

            70%..............15-30 mins (tissue may be left here)

            80%..............15-30 mins

            95%..............30 mins

            100%.............30 mins

            100%.............30 mins

 

5. The tissue may now be transferred to the critical point dryer, but some workers recommend the use of a transitional fluid such as amyl acetate.

 

6. For use of amyl acetate, make dilutions of amyl acetate and 100% ethanol. You will need  30% amyl acetate, 60%, and 90%. Process the tissue for 10 mins each through each dilution, and then through 2 changes (10 mins each) of 100% amyl acetate.

 

                        USE OF THE CPD2 CRITICAL POINT DRYER

 

CAUTION: Do not use this instrument if you are unsure of any of the steps outlined  below. This instrument operates under high pressure which is dangerous for the uninformed user.

 

1. Your tissue should be immersed in alcohol or the transitional fluid. Transfer the specimen to a specimen basket. Put the basket in alcohol or transitional fluid to keep it moist.

 

2. On the CRITCAL POINT DRYER: Check to see that the VENT, DRAIN and FILL valves are closed. DO NOT OVERTIGHTEN THESE.

 

3. Depress the POWER SWITCH and the VALVE HEATER. The buttons should light.

 

4. Depress CHAMBER ILLUMINATION.

 

5. Open the main valve on the CO2  tank slightly to fill the charging hose, then open the FILL valve and watch as liquid CO2  enters the chamber.

 

6. Open the VENT valve slightly. By venting the CO2  you will begin to cool the chamber.

 

7. Observe the  temperature gauge. You will need to experiment with opening the FILL valve and VENT valve alternately filling the chamber 1/2 to 2/3 and then venting to cool the chamber. The pressure readout should be about 800 psi. Use this procedure to cool the chamber to 10-15oC.

 

8. Remove the specimen basket from the alcohol or transitional fluid. Place the basket in the carrier and install the carrier in the chamber. Do not be alarmed if the chamber warms slightly during this procedure. NOTE: If you overtighten the chamber cover at this point it will be extremely difficult to remove at the end of your run.

 

9. If you wish to place transitional fluid in the chamber, consult Dr. Baird or the user's manual.

 

10. Close the VENT valve and open the FILL valve to fill the chamber with enough CO2 to cover the basket(s) you used. Close the FILL valve. Allow the tissue to "soak" in the CO2 for a few minutes.

 

11. Partially open the DRAIN valve and lower the CO2  level to just above the surface of the sample (you will have to estimate this, but the sample has to be submerged at all times). Close the DRAIN valve.

 

12. Open the FILL valve and admit more CO2  into the chamber to cover the basket. Close the FILL valve. Repeat the soak.

 

13. Repeat step 10.

 

14. Open the FILL valve to fill the chamber enough to fully cover the baskets (1/2 to 2/3 full in most cases).

 

15. Close all valves. Remember to close the main valve on the CO2  tank.

 

16. Depress the CHAMBER HEATER switch. The temperature and pressure should begin to rise and the CO2  will appear to boil.

 

17. The pressure will begin to climb to its set point of 1200 psi and the temperature to its set point of 42oC. The machine will probably reach the pressure set point first.

 

18. When a pressure of 1200 psi is reached, the heater light will click off. Open the VENT valve slightly (then close it) to lower the pressure to about 1100, and the light will come back on. Watch the pressure and temperature balance - the chamber is not heating when the pressure is above 1200 and the light is off!

 

19. When the temperature set point of 42oC is reached, the CHAMBER HEATER light will go off. All of the CO2  in the chamber should be gone.

 

20. Open the VENT valve to begin to reduce the pressure in the chamber by about 100 psi per minute. Note that the VENT RATE valve will help you regulate this. It is important to vent slowly to avoid condensation.

 

21. When the pressure is ZERO, turn off the CHAMBER HEATER, VALVE HEATER, CHAMBER ILLUMINATION and then the POWER.

 

22. Open the VENT and DRAIN valves.

 

23. Loosen the cover on the chamber and remove your samples. Note that the samples will be fragile.

 

24. Samples may now be affixed to the stubs using the appropriate mounting medium. If in doubt about this, ASK.

 

25. If you are not going to use the sputter coater, clean up and lock the lab.

 

WHEN YOU ARE FINISHED, BE SURE THAT ALL PARTS ARE RETURNED TO THEIR PROPER CABINETS AND ALL MACHINES TURNED OFF. CLEAN ANY AREAS YOU HAVE USED. FAILURE TO DO SO WILL RESULT IN SUSPENSION OF YOUR SEM LAB PRIVILEGES.

 

LMB 3/99

 

USE OF THE SC4 SPUTTER COATER

 

CAUTION: Do not use this instrument if you are unsure of any of the steps outlined below. This instrument employs high voltage which is dangerous for the uninformed user.

 

1. Open the regulator valve for the argon. NOTE: The main tank valve on the argon tank is always open. The operating pressure on the tank should be 2-5 on the left hand gauge.

 

2. Carefully remove the metal top plate and set it aside. If desired, the glass cylinder may also be removed - again, be careful.

 

3. Place the stubs in the specimen stage.

 

4. Replace the glass cylinder and the top plate.

 

5. Set the timer on the front to the desired setting (if unsure, start with 40 sec).

 

6. Check the GAS ADJUST valve on the side of the chamber to be sure it is closed (clockwise). DO NOT OVERTIGHTEN VALVE, YOU WILL RUIN IT.

 

7. Depress the POWER SWITCH to evacuate the chamber. You will note the pressure drop on the vacuum gauge.

 

8. Open the GAS ADJUST valve to admit argon to the chamber. Allow the pressure to rise to 1-2 mBar and to remain there for 10 sec. Then close the gas adjust valve and allow the vacuum to be reestablished. This is called "flushing". Flush chamber for at least 2 cycles.

 

9. Close the GAS ADJUST valve and pump the chamber to below 0.1 mBar.

 

10. Open the GAS ADJUST valve to the pressure rises to between 0.1-0.2 mBar.

 

11. Depress the READY/TEST switch momentarily and read the discharge on the plasma current gauge. The current should be about 18 mA. NOTE: If the current is too high the needle will arc, and the reset light will illuminate. Press RESET, evacuate some of the argon, and try again. The high current is usually the result of too much argon.

 

12. When the current is about 18 mA you should see a lavender glow in the chamber.

 

13. Depress the START switch to begin sputtering.

 

14. At the end of the sequence, the plasma current will turn off. Depress the POWER SWITCH to discontinue pumping.

 

15. Admit air to the vacuum chamber with the vent valve on the top of the chamber.

 

16. Remove your specimens and replace the top plate.

 

17. Close the argon valve.

 

18. Lock the lab.

 

WHEN YOU ARE FINISHED, BE SURE THAT ALL PARTS ARE RETURNED TO THEIR PROPER CABINETS, AND ALL MACHINES TURNED OFF. CLEAN ANY AREAS YOU HAVE USED. FAILURE TO DO SO WILL RESULT IN SUSPENSION OF YOUR SEM LAB PRIVILEGES.

 

LMB 3/99

 

USE OF THE AMRAY 1810 SCANNING ELECTRON MICROSCOPE

 

NOTE: These basic instructions are intended for individuals who have previously been trained on the use of the SEM. If you have questions at any point, please ask.

 

Basic Lab Rules:

 

  1. No food or drink of any kind (in any container) are permitted in the electron microscope labs.

 

  1. Schedule yourself for time using the sign-up sheets. Be realistic. If you are half an hour late for your time slot, anyone may use the microscope.

 

  1. When Biology 132 is being taught, these students have priority for use and scheduling.

 

  1. Be sure the lab is clean, instruments properly turned off, and the lab locked when you leave. Those who abuse the lab will find themselves lacking access to it.

 

  1. Report any problems immediately to Dr. Baird (SCST 481, x4073). Any description of the malfunction is helpful in diagnosing the problem.

 

OPERATION OF THE MICROSCOPE

 

Use of Automatic Modes:

 

  1. Turn on the air valve (orange) fully counterclockwise.

 

  1. Turn on the water valve (green) fully counterclockwise.

 

  1. Turn on the nitrogen tank - top center valve only, 1 turn is sufficient.

 

  1. Turn on the main switch to the electron microscope. This is located on the back of the left side of the SEM unit. It will take approximately 30 minutes for the diffusion pump to heat to the point that the microscope is ready to use.

 

  1. The scope is ready to use when the diffusion pump light is NOT flashing and the standby light is illuminated. The longer you let the microscope pump at this point, the better it is for filament life.

 

TO INSERT A SPECIMEN:

 

  1.  Depress VENT on the vacuum control panel. When the chamber is vented (about 1.5 mins) open the door carefully, depress STANDBY to shut off the nitrogen gas, and insert your sample.

 

  1. Carefully close the door, remembering the slot mechanism in the back of the chamber.

 

  1. Press EVACUATE on the vacuum control panel.

 

  1. When vacuum is reached the green READY light will illuminate. Wait 1-2 minutes to achieve a good vacuum.

 

TO VIEW YOUR SPECIMEN:

 

  1. Turn the electronic console ON.

 

  1. Increase the kV to the desired setting (10-20 kV is usual). The filament will automatically be brought to saturation.

 

  1. Press AUTO VIDEO, the button will light. You may use the brightness and contrast knobs to make fine adjustments with this mode engaged.

 

  1. Using the X, Y, Z, T, and R adjustments, select the specimen and area you wish to view. These should be moved with care - if a knob does not turn - do not force it!

 

  1. Select the desired magnification using the buttons on the left side of the console. "10" increases or decreases the mag by decades, the double arrows change mag rapidly and the single arrows alter it more slowly.

 

  1. You may now focus the image using either AUTO FOCUS or manual focus with the large knob near the FINAL LENS button. Manual focus is often better. You will probably find it easier to manual focus if you depress the PARTIAL SCREEN button and focus on the image in the box. Depressing the button again will remove the box.

 

  1. Press AUTO STIG to stigmate the image.

 

  1. You are now ready to observe your specimen or to take photos.

 

  1. If you need to leave the microscope for more than 5 minutes:

 

a. Turn the kV off, the red button will illuminate.

b. Press the STANDBY button and do whatever you need to do.

c. When you return, press EVACUATE and wait for the READY light. You may now turn on the kV and resume your observations.

 

TO SHUT DOWN THE MICROSCOPE:

 

CAUTION: These steps must be done in the order listed - don't rush these steps!

 

  1. Turn off the kV until the red light illuminates.

 

  1. Depress the STANDBY button. Depress the VENT button on the control panel.

 

  1. Open the chamber door and remove your sample.

 

  1. Press EVACUATE.

 

  1. Once the READY light is illuminated, press STANDBY.

 

  1. Turn off the main switch on the back of the microscope. Turn of the switch on the wall.

 

  1. Wait 30 minutes.

 

  1. Turn off the nitrogen, air and water.

 

  1. Sign the log book, clean up your materials, and lock the lab.

 

PHOTOGRAPHY:

 

Using the video printer: Make sure the printer is ON. There is a switch on the back of the printer to turn it on and the power switch on the front must also be depressed. When the printer is ready the green icons on the front of the printer will be illuminated.

 

  1. Select the area you wish to record, stigmate and focus.

 

  1. Depress the SLOW SCAN button. The BUFFER button may also be used. When a slow scan image has been obtained, press the print button attached to the video printer. The image should be available in less than a minute.

 

Using the Polaroid Camera: Unless you are in Biology 132, you must provide your own film. Polaroid type 52 gives only prints, Polaroid type 55 also gives a negative if you process it properly.

 

  1. Repeat step #1 above.

 

  1. Depress SLOW SCAN, be sure the scan rate is at 3 or 4. Use the BUFFER if you wish.

 

  1. Move lever on the film back to "L".

 

  1. Insert film and withdraw film cover.

 

  1. Depress PHOTO. This light will flash while the picture is being taken.

 

  1. When the flashing ceases, push the film cover in, switch the lever to "P" and withdraw the film.

 

USING THE PARTICLE MEASURING SYSTEM:

 

  1. Flip the switch that is labeled PMS on the keyboard. An horizontal line should appear.

 

  1. The line may be moved up or down using the Y knob, the X knob will anchor the line to the left, and the X  will allow you to position it to the end of whatever you are measuring. The delta on the screen will give you the measurement. NOTE: If you want this measurement to be very accurate, press the DEGAUSS button prior to measuring.

 

USING THE KEYBOARD:

 

  1. Flip the switch that is labeled KB on the keyboard.

 

  1. A cursor should appear on the screen. You may now use the keyboard to annotate your photo any way you wish.

 

  1. For more features of the keyboard, consult the user's manual.

 

 

 

 

 

LMB 08/03