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A Shortcut Guide for Obtaining Parallel Illumination PDF file

Taken from a procedure written by Richard Gursky of FEI
from the original work of Felix de Haas

March 2009

This procedure is necessary to minimize magnification changes during image focusing.
  1. Carry out all standard microscope alignments including aligning the apertures, doing gun alignment, and minimizing any beam astigmatism. Load in a trusted, saved, alignment file.
  2. Put in a grid with a carbon support film that has 20 nm gold beads on it. A grid of this type is stored in the Polara multispecimen holder in position #5. Correct the eucentric height.
  3. Go to your intended working magnification and carefully focus and stigmate the image. Make sure that Low Dose is off.
  4. Perform the alignments in the Direct Alignments panel specifically including the beam tilt pivot points, beam shift, rotation center, and the coma-free alignments and pivot points.
If these alignments are very carefully performed you should see little change when doing the tests below. If your alignments are off it will be easier to do this procedure first at your working magnification. Make sure the specimen stage has settled down and you have no specimen drift.
  1. Make sure that you are at your working magnification and that your image is focused and stigmated.
  2. Take an image at 5 μm underfocus.
  3. Take an image at 10 μm underfocus.
  4. In Digital Micrograph go to Process/Simple Math.., click the “a+b” radio button, choose your two images and hit OK. If you see movement of the particles like in the image below, the objective lens rotation center is off.
moving beads
Left: Image taken at 5 μm underfocus, screen up magnification is 50,000X. A gold bead is circled. Center: Image taken at 10 μm underfocus. The same bead is circled. Right: Addition of the two images. The movement of the bead is shown in the circled area.
  1. If your rotation center is not too far off go to around 230,000X and carefully re-focus and stigmate your image.
  2. When working on the Sphera the TV camera works very nicely. On the Polara use the Search mode in the Digital Micrograph software. In the Search Mode setup choose continuous capture, gain normalized, an exposure time of 0.3 sec. and a binning of 4. On the right part of the dialog box under CCD Area choose 1/2 CCD. On the Polara this will give you an image size of 512 X 512 pixels. At 230,000 X (screen up value) the size of your box will be 106.43 nm X 106.43 nm. The XY center of the image will be at 53.2 nm,53.2 nm. If you choose any other camera settings go to the Object menu and select Image Display…/Image/Info and you should see the size of the recorded image and make your calculations accordingly. When you move your cursor over the image look down into the Image Status dialog box on the left and it will show you the X and Y positions of your cursor to help you find the image center. It may help to put a small, lab-tape, arrow on the screen at this point (NO china markers or Sharpies on the LCD screens!).
  3. Start the Search image capturing mode. Focus to zero focus and hit the Reset Focus button. Then underfocus to 5 μm.
  4. Find some gold beads and center a bead in the center of the image with the stage shifts. At this magnification accurate stage movement may be difficult. You can change the MF-X,Y buttons so they turn into stage movement buttons. Then turn the stage sensitivity to the slowest setting possible.
  5. Underfocus to 10 μm. Your beads will move off from the center of the screen.
  6. Go to Direct Alignments and start Rotation Center. Turn the amplitude (same as focus step size) to zero (counter clockwise) to stop the focus wobbling and re-center the bead with the MF-X/Y knobs. Turn off Rotation Center.
  7. Go back to 5 μm underfocus and see if the bead moved. Repeat steps 12 through 14 if it moves.
  8. The final step to check the parallelicity of the beam is to repeat this at a different beam intensity. Spread or contract your beam enough so that you can see a definite change. This is illumination I2. Center a bead at 5 μm underfocus. Go to 10 μm underfocus at I2. Is the bead movement more or less than that of the last step? If movement is smaller then change the illumination more in that direction. If greater then back it off. This will be intensity I3.
  9. Check to see if there is any bead movement at I3 when going back and forth between 5 μm and 10 μm underfocus. If there is no movement the beam is parallel. If not, repeat the procedure.
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