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Fei Tecnai_tf20_t12_operating Instructions_v2

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  FEI TECNAI T12 / TF20 Operating Instructions I Start-up II Specimen Loading / Unloading (RT) III TEM Holder Insertion / Removal (RT) IV Alignment of the TEM V Imaging VI End of Session VII “Common” Problems Liguo Wang Department of Biological Structure University of Washington May 2013 [email protected]   29  May  2013     1   Important rules: • • • Column valve should be CLOSED unless you are looking at your sample. No considerable force should be needed for any manual actions on the holder or CompuStage. When the red light on the compustage is on, don’t do any actions on the holder or Compustage. I. Start-up (Do not proceed if these conditions are not met) A) Book the TEM session online. B) Check the logbook about the status of the TEM. If there is any unsolved issue, please do not proceed further and consult with staff. Otherwise, write down your TEM session information in the logbook. C) Fill LN2 cold trap (make sure viewing screen is covered). The first dewar of the day will last approximately 20 minutes. Subsequent dewars should last for a couple of hours. D) Log in to the Tecnai computer using your username and password. Launch the Tecnai User Interface (TUI) and Tecnai Image Analysis (TIA) or Digital Micrograph (DM) programs. E) Check whether the “Vac” and “HT” buttons are lit on the server control panel. If the “Vac” is not lit, do not proceed further and consult with staff. If the “Vac” is lit but the “HT” is not, press “HT” on the server control panel, set it to 120 kV for T12 and 200kV for TF20 in the Tecnai “Filament” window, and click “High Tension” in TUI. Figure  1  TEM  control  console   F) Check that the column valve is closed: TUI è “Setup” workset è “Column Valves Closed” button is yellow. (TF20 in Moles does not have ‘Turbo on’ button)   Figure  2  Vacuum  panel [email protected]   29  May  2013     2   G) Activate “Vacuum Overview” in TUI (lower right portion of the screen). Check to see that IGP 1 vacuum level is 6 (log scale) (Figure  3) or the Gun/Col pressure is 6 in Vacuum workset (Figure  2). If you are the first user of the day, the vacuum may be higher. With the addition of LN2 in cold trap the vacuum will improve.   Figure  3  Vacuum  overview II Specimen Loading / Unloading (RT) A) Keep one hand against the black end cap of the holder, making sure it cannot move out of the cover tube. B) Fit the tool (stored in one of the holes in the supports of the cover tube) into the hole in front of the clamp. Then gently lift the clamp straight up to its fullest extent.   Figure  4  Sample  loading   C) Place the specimen in the circular recess of the specimen-holder tip. D) Gently lower the clamp with the tool straight down onto the specimen. Make sure the specimen remains correctly in position. Return the tool to its stored position. E) Retract the holder slightly in the cover and turn it upside down. Tap the cap at the end a few times. Turn the holder back and check that the specimen has not moved (movement is a sign that it isn't clamped properly). F) Check the O-ring for debris and carefully remove with canned air or a tweezers. Note: Be careful not to touch any part of the specimen holder between the tip and the O-ring with bare hands. Never mount magnetic specimens (disks) in the single-tilt holder. The clamp is normally not strong enough to prevent the specimen from flying out due to the objective-lens magnetic field and sticking to the [email protected]   29  May  2013     3   objective-lens pole pieces. III TEM holder Insertion / Removal (RT) The specimen airlock of the CompuStage and the specimen holder consist of fine, high-quality mechanics. If considerable force is needed for any manual actions on the holder or CompuStage, it is a sign of something being wrong. It should never be necessary to exert strong force and doing so may result in damage to specimen holder or CompuStage. Before both insertion and removal, ensure that the: • Column valve is closed (“Column Valves Closed” is yellow, Figure  2); • Red sample exchange light on the compustage is off; • Stage is at the home position: “Search” worksetè“Stage”è “Control” (flapout tab) èclick “Reset Holder”.   Figure  5  Stage  panel TEM holder Insertion A. Carefully insert the holder, with the holder “orientation and switching pin” (shown in Figure  6) at the 5 o'clock position, as far as it will go into the compustage (be careful not to scrape the tip). Stop and wait for the pumping of the specimen airlock.   Figure  6  TEM  holder  insertion   B. The prepump will start (red light on the compustage will turn on). Select the Specimen Holder you are using in TUI and activate it with the (↵) button. The remaining pumping time for the specimen exchange is shown on the “Vacuum Overview” screen. Usually the displayed time is faster than the red light indicator. [email protected]   29  May  2013     4   C. Stop and wait when the red light is on. Otherwise you may damage the compustage and break the vacuum. D. When the pumping cycle ends (red light on the compustage turns off), rotate the holder counterclockwise from 5 o’clock to 12 o’clock. Do not let go of the holder at this point! E. Gently guide the holder into the column (Don’t let it go by itself) while watching the column vacuum on screen. Tap the end of the holder to ensure stabilization. TEM holder Removal: A. Support the purple surface of the goniometer with left hand. Pull the sample holder straight back with right hand as far as it will go. B. Rotate the holder clockwise from the 12 to 5 o’clock position – i.e. as far as it will go. C. Stabilize the hand holding the sample holder and use the thumb to push the purple plate to release the holder from vacuum. Note: Please be gentle. Gloves are better used. D. Guide the holder out of the column taking care not to scrape the tip along the inside of the goniometer. Or the red light may be lit up. IV Alignment: Always align the microscope from the top down. Begin with the Objective aperture out (Figure  8). Pressing F1 will open a help window to give explanation and assistance at any time. MF: multifunction X &Y; LC = left console; RC = right console.   Figure  7  Left  and  right  control  console A) Generating Emission Current (T12 only) Set the “Heat to” button in “Filament” workset to its maximum value (32) and activate it with the (↵) button. Click “Filament” button to turn on the T12’s W filament (Figure 1). B) Wait for the column vacuum to be smaller than 10 (Figure  2). [email protected]   29  May  2013     5   C) Open Column Valve. Find the beam. If no beam is visible, try the following tricks to get the beam back: 1) The current magnification might be too high. Try to lower the “Magnification” (RC) to find the beam and center it with Beam Shift Track Ball (LC). 2) What you’re seeing may be the grid bar, which holds your specimen. If this is the case, then try moving the specimen with the “Joystick” (RC). Or you could check the beam by pulling the holder out for about 1 cm. 3) It could also be that the beam brightness (“Intensity” knob LC) is too far one way or the other from crossover. Try to turn the intensity knob from one side to the other. 4) The beam might be badly aligned. Try to load a previous alignment file (“Alignment” worksetà flap-out tab “File”àselect the file and apply it). D) Make sure the objective aperture is out. Lower the Magnification to about 390x, search and save the positions of interesting squares. E) Put in the objective aperture, and center it. Objective Aperture Centering 1) Put in the objective aperture.   Figure  8  Aperture  control 2) 3) 4) 5) Change the magnification to about 500X. Center the aperture by adjusting the two screws on the aperture rod (Figure  8). Put in your specimen, and increase the magnification to about 10,000X. Turn on diffraction mode by pressing the diffraction button on RC. Center the weak ring around the bright focused beam as shown in Figure  9 (from the left panel to the right panel) by adjusting the two screws on the aperture rod. Note: a specimen with continuous film is required (an empty region won’t work).   Figure  9  Centering  of  the  objective  aperture 6) Turn off diffraction mode by pressing the diffraction button on RC. F) Find the eucentric height for a square, and focus the image. Eucentric Z height and focus [email protected]   29  May  2013     6   1) Start at low magnification (a few thousands), find a feature and center it using sample-moving joystick (RC). 2) Start wobbler (SearchàStageàControlàwobbler, Figure  5), and adjust Z height (up and down buttons on the RC [These buttons are pressure sensitive. The harder you press, the faster the Z position changes]) to slow down the movement of the feature. 3) Increase magnification, and adjust Z height (up and down buttons on the RC) to slow down the movement of the feature till the feature stays in the view field at 50,000X magnification. 4) Stop wobbler (SearchàStageàControlàwobbler), and go to high magnification (150-200kX). 5) Press Eucentric focus button on the RC, and reset defocus counter (one of the L1-L3 on LC, R1-R3 on RC). 6) Adjust focus knob to find the true focus via the binocular. When you change the focus, you should see a similar image like the following (Figure  13) on the small viewing screen. Tip: put in the beam stopper, and adjust the binoculars to see the clearest edge of the beam stopper.   Figure  10  Finding  the  focus G) Follow the alignment steps (“Alignment” workset) 1) Condenser 2 Aperture Centering (10kX,Spotsize 3) with objective aperture out. a Select an appropriate C2 aperture (e.g. position 3). b Focus the beam to a small spot using ‘Intensity’ knob on LC c Center the beam with the trackball on LC. d Defocus (i.e. spread) the beam with “Intensity” knob (LC). If the aperture is [email protected]   e f 29  May  2013     7   not centered, you will see off-centered distorted beam (left panel in Figure  11). Adjust the two screws on the aperture rod (shown in Figure  8) to center the aperture as shown on the right in Figure  11. Repeat steps b to e to make the defocused beam and focused beam are concentric (right panel in Figure  11).   Figure  11  Centering  of  condenser  aperture 2) “Gun” alignment in “Alignments” workset in TUI without specimen a Gun tilt: maximize the beam intensity (i.e. minimize the measured exposure time) by adjusting MF. b Gun shift (tip: adjust the intensity knob on LC to make the beam a little bigger than the smallest circle on the viewing screen). c Spot size dependent gun shift 3) “Beam HM” alignment in “Alignments” workset in TUI with specimen in a Beam tilt pivot point: minimize beam movement b Rotation center: minimize image (i.e. feature) movement Move a feature to the center of the view field by using the sample moving joystick. Look at the feature on the small viewing screen via binoculars. 4) Condenser Astigmatism via “Stigmator” in TUI Focus the beam at about 100kX (50kX for T12) magnification. If beam is not circular as shown in Figure  12, go to: “Tune”à “Stigmator”à “Condenser”, use MF to adjust the beam roundness. Click “None” after finishing.   Figure  12  Astigmatism  of  condenser  lens 5) Objective Astigmatism via Stigmator in TUI a Put in Objective aperture and center it (Figure  9). b Take a live image at about 50kX-150kX using the search mode of the camera [email protected]   c 29  May  2013     8   (continuous image taking), Do “Live Reduced FFT” (ProcessàLive reduced FFT). If Live FFT image is not circular (left panel in Figure  13), go to: “Tune”à “Stigmator”à “Objective” (Figure  12), use MF to make FFT image circular (right panel in Figure  13). Click “None” after finishing.                 Figure  13  Astigmatism  of  objective  lens V Imaging A) Tecnai Image Analysis 1) Choose BM-Eagle in the CCD/TV Camera panel. 2) Camera Modes: “Search” is a lower resolution, faster refresh rate setting that allows real-time movement about the specimen; “Preview” is a higher resolution setting used for fine focusing and objective astigmatism correction; “Acquire” will capture an image. The only parameter you should/will need to alter is the “Integration Time” for Acquire. Typical vales for bright field imaging range from 12 seconds. 3) To stop “Search” or “Preview”, click “Search” or “Preview” again. 4) Save images by saving data to emi format or exporting data to tiff format. B) Digital Micrograph 1) Ensure the palettes you wish to work with are open on the left side of the viewing screen: “Windows”è“Floating Windows” 2) File Saving 3) Lift the “Viewing Screen” by pressing L1 (LC) 4) Insert the camera: “Camera”è“CCD/TV Camera”è“Insert”. 5) Camera Modes: “Search” is a lower resolution, faster refresh rate camera that allows real-time movement about the specimen; “Preview” is a higher resolution camera used for fine focusing and objective astigmatism correction; “Acquire” will capture an image. The only parameter you should/will need to alter is the “Integration Time” for Acquire. Typical vales for bright field imaging range from 1-2 seconds. 6) To stop “Search” or “Preview”, click on the header bar of the active window and press the “Space Bar”. [email protected]   29  May  2013     VI End of Session A) Column valve closed: Tecnai interfaceè “Set Up” worksetè“Column Valves Closed” button is yellow. B) Stage is at home position: Tecnai interface è “Search” worksetè“Stage”è “Control”è“Reset Holder”. C) “Viewing Screen” is in the down position. D) Retract the holder, and take your sample out. E) Check the online calendar. 1) If other users are going to use the TEM after you, a Fill the cold trap dewar with LN2 for the next user. b Turn off filament (T12 only). 2) If you are the last user signed up for the day a Turn off filament (T12 only). b Turn off the “High Tension” (T12 only). c Remove the LN2 Dewar to let the cryo box warm up. d Start “Cryo Cycle”: “Setup” worksetè“Vac”è“Cryo cycle”.   Figure  14  cryo  cycle e Wait till the process information in “Vacuum Overview” (Figure  3) shows cryo cycle is running. F) Close the TIA or Digital Micrograph and TUI. G) Log out of the Tecnai computer and your session H) Finish your TEM session information in the logbook. VII “Common” Problems • If the left or right console is not responsive, unplug and replug them in • If either the TIA and TUI programs encounter a problem, you may restart them to see if that corrects the problem. Never restart the computer! 9