1.3. Before loading the holder into the TEM, make sure the X tilt is set to zero and the goniometer locked in place (this will make loading easier).

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1 JEOL 200CX operating procedure Nicholas G. Rudawski (805) Specimen loading 1.1. Unlock the TUMI system Load specimen(s) into the holder. If using the double tilt holder, ensure the retention hex ring for each basket on the holder has been screwed in with the correct orientation (threaded side down) Before loading the holder into the TEM, make sure the X tilt is set to zero and the goniometer locked in place (this will make loading easier) Once the X tilt has been returned to zero (the zeros on the left and right dials will line up), pull the short metal cylinder that plugs into the M input on the goniometer inward to lock the goniometer in place Push the holder straight into the load lock lining up the peg on the holder with the slot in the load lock (approximately at 9:00 o clock); continue to hold the sample holder firmly in place and listen for two valves to actuate; the holder may now be released. Wait for two more valves to actuate before proceeding further Turn the holder clockwise until a stop is felt and then gently let the vacuum draw the holder into the column. If using the double-tilt holder, plug in the y tilt cable on the goniometer into the H1 input (there is only one correct way to plug it in). The goniometer can now be unlocked by pushing the short metal cylinder that plugs into the goniometer outward (if specimen tilting is needed) Check the penning gauge on the lower left panel; the vacuum should be < Torr before proceeding (black line). Also, the READY light on the upper left control panel should be lit before proceeding (NOTE: this button can only light up if the vacuum is sufficiently high AND the TUMI system has been unlocked). 2. Generating a beam 2.1. Make sure the objective and selected area apertures are removed. The knobs on the aperture assemblies will be at 0 when the apertures are retracted Under ACCELERATING VOLTAGE on the left panel, press the HT button to turn on the high tension. The 200 button should already be depressed as this is the voltage most often used for operation (this also helps to maintain stability 1

2 of the lenses). Under BEAM CURRENT on the left panel, the needle should jump 5 times as the operating voltage is sequentially increased to 200 kv and stabilize at ~100 µa Uncover the viewing screen and slowly turn the FILAMENT EMISSION knob clockwise until it reaches the mechanical stop (~20 s total time is sufficiently slow). If the filament emission is increased too quickly, this can lead to a blown filament, which will require the microscope to be down for several hours to fix, so take care to increase the filament emission slowly. Some intensity should be evident on the viewing screen (unless something is blocking the beam). 3. Finding a region of interest (if needed) 3.1. Depending on the current position of the holder, the beam will be centered over one of the two specimen positions (remember, each holder can hold two specimens). To translate between the two specimens positions, use the dial located on the left side of the column at the same vertical position as the specimen holder Once the correct specimen position has been determined, find an area of interest on the specimen. If necessary, press LOW MAG under FUNCTION on the right panel to enter low magnification mode. Once an appropriate area of interest has been found, press MAG under FUNCTION to return to regular magnification mode. 4. C2 aperture alignment 4.1. C2 aperture 3 will be sufficient for most imaging situations; DO NOT change the C2 aperture without consulting MAIC staff first; set SPOT SIZE to 1 (middle left panel) and set the magnification to 5k using MAGNIFICATION (right panel) Bring the beam to crossover using C2 (middle left panel); center beam on the viewing screen using COND TRANS (middle left and right panels) Expand the beam (over focus C2) so it fills the viewing screen by turning C2 clockwise; use the aperture shifting knobs on the C2 aperture assembly to center the beam Repeat the previous 2 steps one more time (usually sufficient). 5. Gun tilt/shift alignment 5.1. Bring the beam to crossover using C2 and center it using COND TRANS. 2

3 5.2. Use the TILT knobs under GUN ALIGNMENT (lower right panel) to maximize the brightness of the beam Increase the SPOT SIZE to 3, bring the beam to crossover using C2, and center the beam using COND TRANS Decrease the SPOT SIZE to 1 and bring the beam to crossover using C2 ; use the TRANS knobs under GUN ALIGNMENT (lower right panel) to center the beam on the viewing screen Repeat the previous 2 steps two more times. 6. Condenser astigmatism correction 6.1. Select the SPOT SIZE that will be used for imaging; spot size 2 is recommended for general imaging while spot size 1 is best for DF imaging or very thick specimens. For imaging at very low magnifications, spot size 3 should be used to avoid overexposure of the CCD camera Set the magnification to 10 k, bring the beam to crossover (C2), and center the beam using COND TRANS Slightly de-saturate the FILAMENT EMISSION so an image of the filament (dark ring) is observed in the beam; focus the image of the filament using C Use the X and Y knobs under COND STIGMATOR (lower right panel) to obtain a sharp image of the filament; re-saturate FILMAMENT EMISSION when finished. 7. Eucentric height adjustment 7.1. Reduce magnification to 5 k, bring the beam to crossover using C2, center the beam using condenser TRANS, and turn C2 clockwise to expand the beam to the size of the viewing screen Center a recognizable feature of the specimen on the viewing screen by moving the stage Unlock the goniometer so the holder can be tilted; tilt the holder a few degrees (either way is fine) and note the movement of the feature from the center of the screen; adjust the Z height control until the feature returns to the center of the screen Tilt the holder back to 0 ; Use the X and Y stage translation controls to return the feature to the center of the screen. 3

4 7.5. Repeat the previous 2 steps until the feature remains centered on the viewing screen when the stage is tilted (usually 3 or 4 iterations is sufficient). 8. Deflector coil balancing 8.1. Reduce the magnification to 2 k, bring the beam to crossover using C2, and center the beam using COND TRANS Switch the WOBBLER under COND ALIGNMENT (lower left panel) from the middle (OFF) position to the up (X) position; the beam should split in two Adjust the X CORRECTOR and COMPENSATOR knobs under COND ALIGNMENT (lower left panel) so the two separated beams overlap Flip the WOBBLER switch to the down (Y) position and repeat the previous step using the Y knobs; flip the WOBBLER switch to the middle (OFF) position when finished. 9. Current centering 9.1. Make sure all four OBJ FOCUS knobs are set to the 12 o clock position Increase the magnification to 30 k ; bring the beam to crossover using C2, center the beam using COND TRANS, and then expand the beam so it is bigger than the viewing screen by turning C2 clockwise Find an easily recognizable feature on the specimen (corner of a FIB lamella, distinct particle, etc.) and center it on the viewing screen Turn the coarsest OBJ FOCUS setting counterclockwise from 12 to 9 o clock and note movement of feature of interest from the center of the viewing screen Re-center the feature of interest on the viewing screen using COND TILT Turn the coarsest OBJ FOCUS setting clockwise from 9 to 12 o clock; recenter the feature of interest on the viewing screen by moving the stage Repeat the previous 3 steps until the feature of interest doesn t move from the center of the viewing screen when the coarsest OBJ FOCUS setting changed between 12 and 9 o clock (3 iterations should be sufficient). 10. Obtaining a parallel beam/objective aperture centering Enter SA DIFF (diffraction) mode under FUNCTION (right panel). 4

5 10.2. Insert objective aperture 1 (it is not necessary to shift the aperture at this point); set the camera length to 137 or 205 cm by using the outer CAMERA LENGTH knob under SA/HD DIFFRACTION (right panel) Focus the edge of the aperture using the inner CAMERA LENGTH knob under SA/HD DIFFRACTION (right panel) Focus the spots in the diffraction pattern using C2 (the beam is now parallel) If the specimen needs to be crystallographically aligned, retract the objective aperture (so the whole DP is visible) and adjust the X and Y tilts (the Y tilt uses a foot pedal) to orient the sample; NOTE: if a large Y tilt was introduced, it may be necessary to reestablish eucentric height Insert a smaller objective aperture (2 4) and center it on the direct beam with the aperture shifting knobs (there is no practical reason to use objective aperture 1 for any imaging purpose on this TEM) Return to MAG mode (don t adjust C2!); reduce the magnification until the whole beam is visible and center it on the viewing screen using COND SHIFT (don t adjust C2!) and set the magnification to the desired setting for imaging. 11. Operating in dark-field (DF) mode (if needed) A separate set of deflector coils is used to operate the TEM in DF mode; to activate these coils, select DARK (left panel); notice that the TRANS and TILT knobs under DARK FIELD (left and right panels) are now lit Enter MAG mode (if not already in this mode) and decrease the magnification and/or adjust C2 until the beam is visible; set SPOT SIZE to 1 (best for DF imaging); then use COND TRANS under DARK FIELD to center the area of illumination (NOTE: if you adjust the regular TRANS knobs, nothing will happen as the DF deflector coils are now activated; when finished, select BRIGHT (left panel) to reactive the regular set of deflector coils Enter SA DIFF mode, obtain a parallel beam, and note the position of the direct beam in the diffraction pattern; select DARK to enter DF mode (the diffraction pattern will likely be shifted relative to the diffraction pattern observed with the BF deflector coils activated; adjust COND TILT under DARK so the direct beam (and by extension, the whole diffraction pattern) is coincident with the previous position of the direct beam For a crystalline specimen, tilt so that a two-beam condition is achieved (NOTE: if the Y tilt is adjusted, the eucentric height may need to be reset). 5

6 11.5. To form an CDF image, adjust COND TILT under DARK to bring the direct beam to the position of the Bragg beam; then insert an objective aperture and position it around the initial location of the direct beam (optic axis); make sure the object aperture is sufficiently small (usually apertures 3 or 4) such that only a single beam is included in the aperture. Return to MAG mode and recenter the beam using COND TRANS (don't adjust C2 ) To form a WBDF image, adjust COND TILT under DARK to bring the Bragg beam to the position of the direct beam; then insert an objective aperture and position it around the initial location of the direct beam (optic axis); make sure the object aperture is sufficiently small (usually apertures 3 or 4) such that only a single beam is included in the aperture. Return to MAG mode and recenter the beam using COND TRANS (don't adjust C2 ) When finished operating in DF mode, press BRIGHT to reactivate the original set of deflector coils. 12. Image acquisition Cover the viewing screen; under the Camera View menu in Digital Micrograph, make sure Search is selected under the Setup pull-down menu and input a value of for Exposure (s) (this will be adjusted again shortly) Verify the Camera Inserted and Auto Survey boxes are checked and select Start View to start live imaging Enter the current magnification into the Calibration State dialogue box that pops up; make sure IMAGING is selected under Mode. Translate the stage to find a region of the specimen desired for imaging. 6

7 12.4. Adjust the Exposure (s) time in the Camera View menu to the point where the brightest regions in the image read a few thousand counts (7000 is the theoretical ideal, but as low as 2000 is usually sufficient) Use the OBJ FOCUS knobs to focus the image (slight underfocus) Under the Camera Acquire menu in Digital Micrograph, make sure Record is selected under the Setup: pull-down menu, the Auto Exposure box is unchecked, and input a value ~5 times the value of Exposure (s) used in Camera View for Exposure (s) in the Camera Acquire menu. Select Start Acquire to acquire a still image If image collection at a much different magnification is desired, Exposure (s) in Camera View will need to be adjusted accordingly. As a general rule, the value for Exposure (s) should be doubled (halved) every time the magnification is increased (decreased) one setting to keep the counts similar to the initial magnification setting. Be especially careful when reducing the magnification as it is very easy to quickly oversaturate the camera When finished imaging, select Stop View to stop live imaging and uncheck the camera inserted box under Camera View in Digital Micrograph (DO NOT touch any switches on the MultiScan camera control!); 7

8 13. Acquiring diffraction patterns If the incident beam is still parallel, skip to 13.5 below. Otherwise, perform next three steps Enter SA DIFF mode; use the outer CAMERA LENGTH knob to change the camera length as desired to increase or decrease magnification of the pattern. NOTE: acquired diffraction patterns are only properly calibrated in Digital Micrograph up to camera lengths of 330 cm Insert an objective aperture into the diffraction pattern and focus the edge of the aperture by using the inner CAMERA LENGTH knob (the spots in the diffraction pattern may go out of focus) Focus the spots in the diffraction pattern using C2 ; this ensures that the incident beam is parallel (the calibration in Digital Micrograph will not be accurate otherwise); retract the objective aperture Enter SA MAG mode; use the outer MAGNIFICATION knob under SA/HD DIFFRACTION to change the magnification until the beam and/or sample is clearly visible Use COND TRANS to center the beam on the viewing screen; DO NOT adjust C2 or the beam will no longer be parallel Translate the stage to center the area of interest on the viewing screen, insert selected area aperture 3 or 4 (the two smallest) into the column, and center the aperture on the viewing screen using the aperture shifting knobs (the area of interest, center of beam, and selected area aperture should all be coincident) Enter SA DIFF mode and, if necessary, focus the diffraction pattern using the inner CAMERA LENGTH knob; the beam was already adjusted to be parallel previously, so there is no need to adjust C Turn FILAMENT EMISSION counterclockwise to dim the DP until the direct spot is barely visible on the viewing screen. 8

9 Cover the viewing screen; in Digital Micrograph, make sure the Camera Inserted box under Camera View is unchecked; under the Camera Acquire menu, input a value of 1.0 for Exposure (s) (this will usually be sufficient for acquiring diffraction patterns) Select Start Acquire under Camera Acquire ; select Yes from the dialogue box that pops up regarding insertion of the digital camera; when the Calibration State dialogue box pops up, select DIFFRACTION under Mode, enter the camera length, and then select OK. When the diffraction pattern is done acquiring, immediately uncheck the Camera Inserted check box under Camera View to retract the camera (this prevents further exposure of the camera to the intense diffraction pattern) Return to MAG mode and retract the selected area aperture when finished taking diffraction patterns; resaturate FILAMENT EMISSION. 14. Finishing the session Enter MAG mode: MAG button under FUNCTION Reduce magnification to Set SPOT SIZE to Turn C2 all the way counter clockwise Set the FINE and MEDIUM OBJ FOCUS knobs to the neutral position (for stability of the lenses in the microscope) Return the X tilt to zero by manually adjusting the goniometer so the two zeros on the two dials line up. If using the double tilt holder, return the Y tilt to zero by adjusting the foot pedals so the 0 reading on the holder is lined up with the red line on the gauge; unplug the Y tilt cable from the H1 input on the goniometer (again, if using the double tilt holder); lock the goniometer in place as described in the loading section On the SPECIMEN POSITION indicator (left panel), adjust the specimen translation wheels so the indicated position (small bright dot) is centered (helps to prevent holder damage during unloading) Retract the objective and selected area apertures; do not retract the C2 aperture Slowly turn the FILAMENT EMISSION knob counter-clockwise back to zero. 9

10 Leave the 200 button depressed under ACCELERATING VOLTAGE (again, this helps to keep the lenses stable) and press the HT button to turn off the high tension; the holder is now ready for extraction Pull the holder straight out until a stop is felt, then counter-clockwise until another stop is felt, then straight out again Remove the sample from the specimen holder and cover the end of the specimen holder with the protective plastic cover; if using the double tilt holder, return the retaining hex rings and hex ring tool to the appropriate containers Log off the TUMI system. Appendix A: shorthand alignment/imaging instructions 1. Find region of interest (use LOW MAG mode if needed, but then return to MAG mode) 2. Align C2 aperture 3. Align gun tilt 4. Align gun shift 5. Select spot size to be used for imaging 6. Correct condenser astigmatism 7. Set eucentric height 8. Adjust shift purity (deflector coil balancing) 9. Adjust beam tilt (voltage centering) 10. Enter SA DIFF mode, obtain a parallel beam, and insert objective aperture and center around direct beam 11. Return to MAG mode and center beam (do not adjust CONDENSER knob) 12. Set magnification to desired setting 13. Insert camera and start acquiring a live image 14. Focus image and adjust exposure time until image is properly exposed 15. Using an appropriate exposure time for acquisition mode Appendix B: shorthand instructions for acquiring selected area diffraction patterns 1. Make sure the basic alignment has been performed 2. Obtain parallel beam (if beam not already parallel) 3. Enter SA MAG mode and adjust magnification in SA MAG mode as needed; center beam on viewing screen 4. Center area of interest on viewing screen with stage controls 5. Insert SA aperture 3 or 4 6. Center SA aperture on area of interest and return to SA DIFF mode 7. Make sure objective aperture is retracted 8. If needed, focus diffraction pattern using diffraction focus (not C2) 9. Dim DP by turning FILAMENT EMISSION counterclockwise until direct spot is barely visible on the viewing screen. 10. Go straight into acquisition mode and acquire a diffraction pattern (do not acquire a live diffraction pattern) with 1.0 for Exposure (s) 11. Immediately retract the digital camera when finished 10

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