Operating Procedures for TEM2 FEI Tecnai

Note: Do not press the buttons on the TEM. This will turn the TEM off and will take hours to bring it back up. Please do not reboot the TEM computer. This will turn off the vacuum and the high tension. If you are having problems with the TEM, please find a SMIF staff member immediately.

1) Log usage into the SMIF web site. 2) Check to see if the camera boxes are on. a) The TIA camera box is the box behind the monitors. The switch will be lit green if on. b) The AMT camera box is on the floor under the Cryo holder. The green light above the switch should be on. c) If either of the cameras were off, please find a SMIF staff member to turn on. The cameras must cool down for 1hr before they can be used. 3) If needed, log into the TEM computer. User name is : TEM Users, Password is: tecnai 4) If needed, open the Tecnai User Interface and then open the TIA software. If the TIA software crashes, please find a SMIF staff member. 5) Place LN2 into the cold finger dewar. The LN2 needs to be topped off before each use.

Turning on TEM

Note: It is important to change the kV before ramping up the Heat to # when turning on the TEM.

1) There are tabs at the top left of the Tecnai software. Go to the Tune . Under the Control Pads box click on Fluorescent Background Light . This turns on the lights on the control panels. 2) Press the Set up tab and look for the Vacuum box. 3) The (at top of the box) should read COL. VALVES. If it reads CRYO CYCLE or COL. COND, a cryo cycle is running and it has to complete before the TEM can be used. If there something other than the above or READY in the Status bar the TEM is not operational. Please contact a SMIF staff member. 4) Check the vacuum readings in the Vacuum box. The Gun should be less than 25 and the Column should be less that 15. If the values are great than this, please wait till they return to these values before continuing. 5) Check the High Tension setting (kV) and check to see if the High Tension button is yellow. If not yellow, press the button to turn on. a) If the button does not go yellow, check to see if the HT button on the TEM is lit. If it is not lit, press it to turn on. If it is lit, try pressing the High Tension button in the software again. If it still does not go yellow, find a SMIF staff member. 6) Change the kV if needed, using the pull down . 7) Adjust the Heat to # to the setting for filament saturation at your working kV (these are posted on the TEM). Press the enter button next to the Heat to #. The filament takes about 15mins to warm up from a Heat to # of 15 and 40mins if it was off. 8) Adjust Step # to the setting for the working kV (these are posted on the TEM). 9) Check the Filament button to see if it is yellow. If it is not, press it and it should turn yellow. 10) Adjust the Spot size to 3. 11) The emission current should read between 4-8 µA once the filament has reached the desired Heat to #. The emission current can be increased by changing the Step # one number at a time. 12) Press the Col Valves Closed button (button will turn grey). This will unblank the beam. TEM2 Operating Procedures Revision 6 Page 1 M. Plue 05/31/2017 13) Reduce the size of the beam with the Intensity knob on the left panel. Then bring the beam to the center of the screen with the track ball (also on the left panel). 14) Spread the beam with the Intensity knob so that it covers the entire viewing screen.

Gain Reference with the TIA Camera

Note: A gain reference should be acquired before imaging your samples. It needs to be completed at least once a day.

1) A sample should not be in the way of the beam. This includes films on a grid. 2) Adjust Exp time to 7-8 sec using the Intensity knob (Exp time is found on the bottom right of the TEM software). 3) Raise the viewing screen by pressing the R1 button on the right panel and place cover over viewing . 4) Go to the CCD/TV tab and press the Preview button. 5) Press the arrow button at the top of the CCD/TV box. This will give you another menu screen. 6) Under the Bias/ Gain tab, press the All Gain button. 7) Click Yes in the on the image screen. The gain reference will automatically be acquired. 8) A text box will display on the image. When the gain reference is finished, please close the box. 9) Press R1 and lower the screen. 10) Place sample into the column (see instructions below).

Gain Reference with the AMT Camera

Note: A gain reference should be acquired before imaging your samples. It needs to be completed at least once a day.

1) A sample should not be in the way of the beam. This includes films on a grid. 2) Adjust exposure time to 3 sec using the Intensity knob. 3) Lower the viewing screen on the TEM by pressing the R1 button. 4) Press the scroll wheel and the right mouse button to toggle the mouse and keyboard to the AMT camera monitor. 5) Open the AmtV600 . 6) Press the Camera Out button to insert the AMT camera. 7) Press the Click for Live Image to view an image. 8) Select Corrections on the top . 9) Select Acquire a Background and then press the Proceed button. 10) When the gain reference is completed, remove camera by pressing the Camera In button. 11) Place sample into the column (see instructions below).

Removing and Placing sample into TEM

Note: Wear gloves when handling the sample holder. Never touch the gold part of the sample holder, even with gloves on. At the end of the session, replace the sample holder into the TEM.

1) Press the Set up tab and got to the Vacuum box. 2) Close the Column Valves by pressing the Col Valves Closed button (button will turn yellow). 3) Check the position of the sample holder. The x & y values are posted at the bottom right of the TEM software. If it is not at 0x & 0y, the stage should be returned to this position.

TEM2 Operating Procedures Revision 6 Page 2 M. Plue 05/31/2017 a) To do this, go to the Stage tab and then go to the Stage box. Look for the arrow button. Press it and another Menu box will be displayed. b) Under the Control tab, find the XY button. Pressing the XY button will return the stage to 0x, 0y. 4) Return to the Set Up tab and note the vacuum values. 5) To remove the sample holder from the TEM, complete the following steps: a) Place one hand on blue/purple plate and grasp the handle of the sample holder with the other hand. Pull the sample holder slowly straight out of TEM until it stops. Do not let go or the sample holder will be pulled back into the TEM. This can damage the sample holder. b) Turn the holder half a turn clockwise and wait 10 seconds. c) Pull the sample holder straight out of the TEM. 6) Place the sample holder into the protective receptacle. Remove the end cap. 7) Raise the sample clamp with the pin. 8) Remove grid (if necessary) and place a new grid in the sample holder. 9) Lower the sample clamp with the pin and remove sample holder from receptacle. 10) To place the sample holder into the TEM, complete the following steps: a) Line the small post on the sample holder to 12 o’clock and the larger post on the handle to 6 o’clock. b) Slowly place the sample holder into the sample exchange until it stops. The red light will come on and the pump will start. c) Turn the sample holder ½ a turn clockwise. You will feel the sample holder slip into place. d) Select single tilt holder on the bottom menu bar and press the return button. e) Wait for the Status bar to change from Airlock to Col Valves. The red light on the stage will go off and the pump will stop. f) Rotate the sample holder counter-clockwise about a half turn. g) Insert the holder into the TEM slowly. Be careful not to let the holder be pulled into the TEM. 11) Check the Column pressure and Gun pressure (in the Vacuum box). If the Column pressure is greater than 15 log and/or the Gun pressure is greater than 25 log, wait until they return to 15 and 25 before opening the Column Valves. 12) Open the Column Valves by pressing the Col Valves Closed button (button will go gray). 13) If you do not do not see the beam, complete the following steps: a) Go down in magnification and move the stag (with joystick on right panel). b) If you still do not see the beam, turn the Intensity knob (on the left panel) both ways and move the beam (with the track ball on the left panel). c) If you still do not see the beam, remove the Objective aperture. d) If you still do not see the beam, find a SMIF Staff member.

Note: If the Column, Camera and/or Gun Pressure read 99 and there is a red line through it, please contact a SMIF Staff Member immediately.

Condenser Aperture Alignment and Stigmation Correction

Note: The condenser aperture is the top aperture. Normal setting is 4; for diffraction use a setting of 3.

1) Go up to about 22kx in magnification and reduce the size of the beam using the Intensity knob on the left panel. 2) Bring beam to the center of the screen with the track ball. 3) Increase size of the beam by turning the Intensity knob clockwise. Fill about ¾ of the screen with the beam. 4) Center the beam with the knobs on the condenser aperture. 5) Decrease size of beam to fill about the small circle on the screen.

TEM2 Operating Procedures Revision 6 Page 3 M. Plue 05/31/2017 6) Go to the Tune tab and select the Condenser button on the Stigmator box. 7) Use the x & y multifunction knobs to make the beam as small and as round as possible. a) Check the roundness of the beam by turning the Intensity knob clockwise and then counter- clockwise. 8) When finished, click the None button.

Objective Aperture Alignment

Note: This is the middle aperture. The bottom pin should be placed to the left to insert the aperture into the column. Normal setting is 4.

1) Bring some of the sample onto the viewing screen using the joystick. The objective aperture alignment needs to be completed with the beam on the sample. 2) Bring beam to the center of the screen with the track ball. 3) Go up to about 10kx in magnification and increase size of the beam to fill about ¾ of the screen with the Intensity knob on the left panel. 4) Press the Diffraction button on the right panel. This will help align the objective aperture. 5) Decrease the size of the bright spot with the Intensity knob. 6) Use the knobs on the objective aperture to center the bright spot within the light green halo. 7) When centered, press the Diffraction button again to return to a normal image.

Focusing the Z axis

Note: The Z needs to be corrected each time a sample is placed into the TEM.

1) Find a landmark on the sample and bring the magnification to about 10Kx. 2) Expand the beam to cover the entire viewing screen. 3) Under the Stage tab, in the Stage2 box, there is an arrow button. Press it and another Menu box will be displayed. Under the Control tab, find the Wobbler button and press it. 4) Pressing the Wobbler button will cause the stage to rock back and forth. 5) Use the z axis buttons next to the stage joystick to decrease the amount of image shift. a) When focused, the image will pulse in and out. b) The Z value is located in the bottom text window under the X an Y readings. 6) Press the Wobbler button again to stop the stage movement.

Imaging Samples with the TIA Camera

1) Center the sample on the viewing screen with the joystick. 2) Spread the beam, with the Intensity knob, until the Exp time (on the bottom of TEM software) reads about 7-8 sec. a) The Exp time is read from the viewing screen. It is only accurate when the viewing screen is down. b) When going up and down in magnification, the Exp time will change, but the reading on the software will not. c) The image from the camera will get gray when the Exp time is too high and very bright when the Exp time is too low. Having the Exp time too low can damage the camera sensor. d) To readjust the Exp time, lower the viewing screen with the R1 button. Then adjust the Exp time back to 7-8 sec with the Intensity knob.

TEM2 Operating Procedures Revision 6 Page 4 M. Plue 05/31/2017 3) Raise the viewing screen by pressing the R1 button on the right panel and place cover over viewing window. 4) Press the Preview button under the CCD or Stage tab in the CCD menu box. This will bring up the image on the 2nd monitor. 5) Adjustment to focus can be made using the Focus knobs. a) Start focusing at a lower magnification. Move to an area on the sample to focus. Move stage with the joystick on the right panel. b) Focus the sample with the focus knob on the right panel. c) The lower knob sets the focus step (on the bottom of TEM software). The smaller the #, the finer the focus. d) The top knob focuses. e) At high magnifications (over 280kx) remove the objective aperture. f) The objective stigmation may need to be adjusted at magnifications over 100kx. i) Go to the CCD menu box. Press the Search button; this will digitally zoom in on the image. ii) Press the Live FFT button, found below the Acquire button in CCD menu box. This will give you another display box on the camera monitor. The stigmation can be corrected using the FFT image. iii) Under focus the image until the FFT image expands out. iv) The objective stigmation correction is found under the Tune tab, in the Stigmator box. Press the Objective button (button will turn yellow). v) Using the x & y multifunction knobs adjust the FFT image until the outer ring is round. vi) When finished, click the None button in the Stigmator box. Then press the Live FFT button to turn off the FFT image. vii) Press the Preview button to continue viewing the sample. viii) The focus may need to be adjusted after completing the objective stigmation correction. 6) To image sample, press the Preview button again (this turns Search off) and then press the Acquire button. 7) To save image, right click in the image. 8) Select Export Data from the menu. 9) Save image to “User Files 2 on SMIF TEM”. a) This folder is found under Entire Network. b) This folder is on the computer next to the TEM. c) Users may make folders under this folder. d) Do not save images to the TEM computer. 10) Follow widows naming procedures to name your images. 11) Select “pc tif w/scale marker” to save images with the scale bar. 12) Press the Save button. 13) To close the image, go to File on the menu bar and select Close. Do not save the changes. 14) Press the Search button again to continue viewing the sample. 15) When finished viewing a sample, press the Search button to stop the camera. Then lower the view screen by pressing R1. 16) Images are saved to the SMIF login computer. The User name is Supervisor, and the Password is tecnai. To remove your images: a) Click on Shared Documents icon b) Open User Files 2 and then open your folder. c) Remove your files. This computer has USB ports in the front and a CD writer.

TEM2 Operating Procedures Revision 6 Page 5 M. Plue 05/31/2017 Imaging Samples with the AMT Camera

Note: There is no password on the AMT computer.

1) Lower the viewing screen on the TEM with R1. 2) Spread the beam until the Exp time (on the bottom menu bar) reads about 3.0s. a) The Exp time is read from the viewing screen. It is only accurate when the viewing screen is down. b) When going up and down in magnification, the Exp time will change, but the reading on the software will not. c) The image from the camera will get gray when the Exp time is too high and very bright when the Exp time is to low. Having the Exp time is to low can damage the camera sensor. d) To readjust the Exp time, press the Camera In button to remove the camera from the TEM. Then adjust the Exp time back to 3.0 sec. 3) Press the scroll wheel and the right mouse button to toggle the mouse and keyboard to the AMT camera monitor. 4) Open the AmtV600 icon. 5) Press the Camera Out button to insert the AMT camera. 6) Press the Click for Live Image to view an image. a) The 4x4 survey is TV rate. 7) Adjustments to focus can be made using the Focus knobs. a) Start focusing at a lower magnification. Move to an area on the sample to focus. Move stage with the joystick on the right panel. b) Focus the sample with the focus knob on the right panel. c) The lower knob sets the focus step. The smaller the #, the finer the focus. d) The top knob focuses. e) At high magnifications (over 280kx) remove the objective aperture. f) The objective stigmation may need to be adjusted magnifications over 100kx. i) The stigmation can be corrected using the FFT in the camera software. This button is found under the Search button in CCD menu box. ii) Under focus the image until a ring is seen around the FFT. iii) Adjust the stigmation using the x & y multifunction knobs until the outer ring is round in the FFT image. iv) When finished, click the FFT button again to turn it off. v) It is easier to use the TIA camera to adjust the objective stigmation. See above instructions. 8) Press Click for Final Image to capture an image. 9) To save an image, go to File and Save as. Choose Save with Caption to save the scale bar. 10) To measure particles, select Proc/Meas from the menu bar. Select Point to Point Measurements. 11) When finished viewing a sample, press the Abort button to turn off the camera. 12) Press the Camera In button to remove the camera from the TEM. 13) Close the AMT software when you are finished. 14) This computer is on the right side of the TEM. There is a USB port to download your images.

Diffraction

Note: The AMT camera should be used for imaging diffraction patterns.

1) Select the area of interest. 2) Set Condenser aperture to 3 and align if necessary (see pg 3). 3) Remove the Objective aperture. 4) Spread the beam to cover the entire viewing screen.

TEM2 Operating Procedures Revision 6 Page 6 M. Plue 05/31/2017 5) Place the Selected Area Aperture into the column (optional). 6) Press the Diffraction button. 7) Insert the pointer to blank out the bright center of the diffraction pattern. 8) Focus the diffraction pattern with the focus knobs. 9) Insert the AMT Camera and image the Diffraction pattern (see above AMT camera instructions). 10) To measure Diffraction pattern rings, select Proc/Meas from the menu bar. Diffraction Measure will be under the Select Measure Mode.

Procedures for Leaving the TEM for the Next User

1) Go to the Set Up tab. In the Vacuum box, close the Column Valves by pressing the Col Valves Closed button. 2) Reduce the Heat to # to 15 and press the Return button. 3) Reduce the Step # to 1. 4) Lower the view screen by pressing R1. 5) Make sure the camera software is turned off (see TIA or AMT camera instructions above). 6) Remove your sample from the TEM (see pg 2). 7) Replace sample holder entirely into TEM (see pg 3). 8) Change the kV to 160 (make sure the Heat to # is15 before changing the kV). 9) Go to the Tune tab. Under the Control Pads box click on Fluoresces Background Light button to turn the panel lights off. 10) Check SMIF calendar to determine if you are the last user of the day. If this is the case: a) Turn the filament off by pressing the Filament button. b) Remover dewar from the cold finger. c) Empty any LN2 into Styrofoam cooler and place dewar upside down in plastic tray. d) Place plastic container under the cold finger. e) Go to the Set up tab and press the arrow button in the Vacuum box. f) Press the Cryo Cycle button under the Cryo tab (make sure the Heat to # is15 before starting the Cryo Cycle). g) Button will turn yellow when activated. The Status bar under the Vacuum box will turn red and read Cryo Cycle. 11) If the next user will not be in for 4 or more hours, please top off the liquid nitrogen dewar. 12) Shrink the TIA and Tecnai software. 13) Logoff usage on the SMIF web site.

Operating Notes

 Apertures – higher #, larger aperture  Apertures are in if pin is pointing left and out if the pin is pointing right  Bottom aperture is the Selective Area Aperture. It is used for Diffraction.  Spot size – smaller #, larger spot  Focus steps – smaller #, finer focus  Stigmator steps - smaller #, finer adjustments  Low Magnification and High Resolution imaging - remove the objective aperture.  Biological samples - set the objective aperture to 4, condenser aperture to 4.  To get more “light” - use a larger objective aperture and a small spot size.

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