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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 The Tecnai F20 cryo electron microscope is a high resolution electron microscope that can be operated at 120 or 200kV. This microscope is equipped with a Field Emission Gun (FEG) as its illumination source and is ...
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limited storage space and they are not for longterm data storage. Two pieces of software control the microscope and digital camera, the Tecnai User Interface which connect ...
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icons. 5. Check that the H igh Tension (HT), O perate and P ower buttons are lit (yellow) on the Tecnai User ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 9. Fill the liquid nitrogen dewar. You'll need to fill it 2x initially. The first time a lot of LN2 will quickly ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 II. Room temperature (RT) holder insertion: The compustage: The compustage is composed of the airlock and the stage itself. The stage is computer controlled (can move ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 Procedure : During specimen insertion and retraction you should n ot need to use considerable force to load ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 7. Carefully place your grid in the opening on the tip. Make sure it resides in the circular recess of ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 11. Carefully insert the holder in this position, then turn to the 5 o’clock position (this aligns the pin ...
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engaged, do not push, jam or otherwise force the holder into the compustage. 13. Select Single Tilt Holder on the Tecnai User Interface. 14. There will be a wait time of 6090s while the airlock is evacuated. 15. Watch out for when the red light turns off and the counter on the User Interface reaches 0s. 16. Securely turn the RT holder from the 5 o'clock position to the 12 o'clock position and gently ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 17. Make sure the black part of the holder is flush with the compustage. 18. Turn off the Turbo pump. 19. Wait ~10 minutes to open the column valves, so as to give time to the stage to stabilize ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 III. Alignments • The purpose of the alignment of the microscope is to ensure that the beam is parallel to the column and that the apertures are properly centered. • Alignments should be done before every imaging session. • Only load the most recent alignments file, made by the FEI engineer. Do not save your own alignment file. It will be deleted. The recent alignment made by FEI will only need to be touched up and it's a good place to start. 1. DO NOT DO ALIGNMENTS IN LOW DOSE 2. Make sure that “Low Dose” is turned off (the box will be gray) 3. Remove the Objective Aperture : Move the small pin on the Objective aperture to the right or to where the point is pointing to the specimen holder. This allows you to do an alignment without the objective interfering 4. Add a spacer to the sample holder in the compustage. * this must be done with the column valves closed! The spacer is typically a BIC pen cap. This spacer allows for the sample to be removed from the beam path without breaking the seal of the sample chamber. The spacer makes the alignment process easier. 5. Wait for the column vacuum to recover after inserting a sample. This takes ~10 minutes. You want IPG1=6 and IGP4=1. These values can be found in “Vacuum Overview.” This is displayed on the right side of the left computer monitor (If it is not there then it can be selected in the bottom right corner of the left computer monitor). 6.
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 12. Select C2 aperture: Determine which C2 aperture to use (as of 3/28/16). Position 1 = 50 Position 2 = 70 Position 3 = 100 *most commonly used for negative stain Position 4 = 150 13. Center the Condenser aperture (C2): Using the intensity knob, condense the beam and go through crossover and monitor that the beam spreads evenly about the center. If adjustments are needed, center the condenser aperture (C2manually) by very slightly turning the center and leftside knobs of the C2 Aperture until the beam is centered on both sides of crossover. 14. Check that the beam is still round. If not follow the procedure outlined in step #11 15. Gun Tilt: Lower the magnification to ~10,000x. To adjust the gun tilt (minimize exposure time) go to the Tune tab and select “Gun Tilt” under Direct Alignments. Bring the large screen down, move the small screen into the beam path (physical tab to the left of the microscope). Using the multifunction knobs (set to gun tilt) adjust the brightness. Keep an eye on the exposure time (“Meas. Exp.” on the monitor) to make it as low as possible. Click done when finished with alignment. Move the small screen back to its original position (out of the beam path). 16. Gun Shift : Perform the gun shift in Alignments. Click on Gun Shift and follow the directions. The magnification and spot size will be set automatically. The idea behind this alignment is to condense the beam and using the multifunction X and Y knobs to center the beam. Follow the instruction in the left hand panel in the FEI user interface. In order to get a more accurate alignment be sure to click “Normalize All” ( typically set to L2 on the left control pad) before and after each step. Repeat the ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 22. Select Objective aperture : Determine which objective aperture to use (as of 3/28/16). Position 1 = 10 Position 2 = 20 Position 3 = 40 *most commonly used for Negative stain Position 4 = 100 23. Diffraction: If the objective aperture is not inserted, insert it to the appropriate setting (if the small pin is pointing to the right/toward the specimen holder then the objective is out, if the small pin points to the left the objective is in). Click on the Diffraction button on the right control panel and condense the beam to a small point. Verify that the aperture is centered appropriately by observing the halo around the small point (it should be even and continuous). Adjust as needed (usually very minimal change) and exit diffraction mode by clicking the diffraction button. 24. Check C2 stigmation: Condense the beam, go through crossover and verify that the beam is round and even. Correct if needed by going to Tune and select Condenser. (To ensure that you can get back to where you started right click on the highlighted settings and copy them to another panel ie. “copy 3 to 1”) Use the multifunction knobs to adjust the beam to make it round. Click d one when finished 25. Check objective stigmation (done with the CCD): Find focus (can be done using the Live View on Digital Micrograph or by inserting the small screen and adjusting the focus until all contrast has ...
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13. Return the next day to retrieve Data. Data will be moved overnight to the computer located behind the curtain in the F20 room V. Imaging 1. Open Digital Micrograph 2. Set exposure time to 1 second 3. Want the mean value to equal ~1000 ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 VI. Sample Loading (cryo) 1. Make sure the anticontaminator dewar is filled with liquid nitrogen (LN2). 2. The cryo holder should be pumped at least 2hrs in the Dry Pumping Station (see separate ...
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CSB CryoEM Facility TF20 Operating Instructions SEC 05/02/16 9. Cool the large forceps and transfer the grid box into the position in the transfer station. Cool the ...
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about 1 inch further. 19. Select the ST Cryo holder option (bottom of the Tecnai User Interface). 20. Wait for the airlock pumping cycle to finish (6090s) as indicated by the timer on the UI. 21. When the holder is upright, reset the stage tilt to 0° by going to the Search tab, flapout window, ...
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there on how to warm it and pump it properly. 25. Close Digital Micrograph and the Tecnai User Interface and log out. 26. Please sign up on the user's sheet. Record the actual time spent imaging. 27. Return the next day to retrieve Data. Data will be moved overnight to the computer located ...
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