User Instructions for 3200FSC

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• Slit to control inssertion of Energy Filter

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". A new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• Tilt grid to 15 degrees
• If you are not eucentric, you will see image shift on the live display camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times

1. Go to Focus, then MDS photoset
2. Lower screen
3. Use specimen movement to center the object manually
4. Go back to Search mode
5. Be sure that PLA button is lit
6. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Do not worry about astigmatism in focus mode. Becasue of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try recentering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Press Low Mag button and lower mag to 150X
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit EM Menu 3 if it is running
5. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
6. Set up Record at binning 4, and 0.1 second exposure
7. Take a test record image to be sure exposure is good
8. Open a new Navigator window
9. Choose to make a full grid montage. Change overlap to 15% and choose to ignore pieces to skip.
10. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
11. Once done, choose to make a new map.
12. If map doesn't look good, try turning off or on "sloppy montage" option and see which is better. Double click map to reload
13. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag
14. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
15. Optionally, save navigator file
16. Quit SerialEM, go back to mag 1 mode
17. Cycle through hysteresis cycle a few times. You will likely need to realign things.
18. Restart EM Menu 3 if you were using it
19. In Search mode, go to saved positions and search there

SerialEM

• Do not use energy filter unless you are in SerialEM low-dose mode
• Never let SerialEM adjust eucentricity in low-mag mode
• For montages, use at least binning 4
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)
• If using energy filter, center it at the same mag that record will be taken at
• To program SerialEM to use Energy filter, go to Record mode, insert slit, click then unclick "Continuous update of mag and beam"
• If energy filter is properly centered, should get around 2 um of image shift at 20K mag before black spot appears
• In tilt series, limit image shift to the maximum you have before a black spot appears (around 2um)
• The smaller the slit width, the more difficulty you will have keeping SerialEM aligned. 20 eV is recommended

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007