Dual Tilt axis tomography on the JEOL-3200FSC

• The JEOL-3200FSC comes with two sets of cartridges: single and double tilt
• The double tilt cartridges show a smaller area of the specimen, ~1mm in diameter.
• The spacer and the clip ring are in one single piece in these cartridges.
• The same tool is used for loading the cartridges and loading is done in a similar way.

1. When the cartridge is loaded, it will be in a configuration such that it can only be rotated counterclockwise.
2. Do a low mag montage or whatever you need to recognize the same area of interest afterwards.
3. Go to the area of interest to collect the first tomographic series.
4. Mark the current location on the JEOL software, by clicking on the spc window.
5. Reset the holder to the zero position.
6. Put the "L" cartridge on the holder that says "jig holder" and insert it into the chuck (after cooling it!)
7. Once the jig is in the chuck, place the chuck spacer so that the jig stops at the proper location
8. Retract the jig and remove it from the chuck
9. Click on the spc window the icon for rotation of the holder
10. Click the "recall" button. This will take you to a place close to the area of interest.
11. Re-identify area of interest and proceed to data collection.

• Set ALLOWTOPICVIEW =

-- RubenDiaz - 31 Mar 2009

Dual Tilt axis tomography on the JEOL-3200FSC

New - How to use the tilt holders of the 3200

• The JEOL-3200FSC comes with two sets of cartridges: single and double tilt
• The double tilt cartridges show a smaller area of the specimen, ~1mm in diameter.
• The spacer and the clip ring are in one single piece in these cartridges.
• The same tool is used for loading the cartridges and loading is done in a similar way.

• Set ALLOWTOPICVIEW =

-- RubenDiaz - 31 Mar 2009

Dual Tilt axis tomography on the JEOL-3200FSC

New - How to use the tilt holders of the 3200

• The JEOL-3200FSC comes with two sets of cartridges: single and double tilt
• The double tilt cartridges show a smaller area of the specimen, ~1mm in diameter.
• The spacer and the clip ring are in one single piece in these cartridges.
• The same tool is used for loading the cartridges and loading is done in a similar way.

1. When the cartridge is loaded with a grid, the natural position will leave the grid in such a position that it can be
2. rotated counterclockwise.
3. This is achieved with the rotation tool that is labeled "L"

• latest version of serialem has a new mode, "search" at left edge of low-dose control panel
• search mode is ONLY for searching -- there is no associates camera mode
• set up as follows

1. Go into Tecnai low-dose as normal.
2. Go to exposure mode. You will stay there your entire session.
3. Set up View, Focus, Record, Trial as normal.
4. Choose "Search" as area to show when screen down.
5. Take a "View" picture.
6. Lower screen.
7. Go to diffraction mode. Choose spot size 9, D 1.35 M (screen is down).
8. Check and uncheck continuous update of mag and beam (to save these settings).
9. If beam is not in center, diffraction needs to be aligned as folows.
   1. Be sure Continuous Update of Mag and Beam is unchecked.
   2. Go to Tecnai Tune, under Image HM-TEM choose Align Diffraction Pattern.
   3. Your settings will change. Raise then lower screen using Screen lift on microscope (NOT R1).
   4. Using MultifunctionX-Y, move beam over approximate position of off-axis camera. May need to lower distance to Trash.findDFdf beam.
   5. Raise screen, move toggle to TEM mode, and unblank beam using SerialEM unblank button.
   6. Using multifunction X-Y knobs, center feature of interest to match "view" picture.
   7. Note that Fastscan camera settings should be "No Mirror" "Rotate right 90" to match SerialEM.
   8. Click "Done" under Tecnai alignment when finished.

Instructions for using this mode

• To go to search, be sure that "Search" radio button is highlighted as mode to go to when screen is down
• Lower screen.
• Switch toggle to TEM.
• Raise Screen.
• Unblank beam.
• To go back to view, switch toggle to CEM and take a view picture.

New - Using SerialEM search mode rather than JEOL MDS

• As with Tecnai, latest version of SerialEM understands diffraction mode and has a Search area in low-dose mode
• Set up is as follows:
• Set up View and Record as you normally would.
• If you want to do a live focus rather than let SerialEM do it, set up focus as follows:
  1. Select Focus under Area to show when screen down, then lower screen.
  2. Change magnification to ~200K, adjust brightness and spot size as desired.
  3. Check then uncheck Continuous update of mag and beam to save above parameters.
  4. Check Additional beam shift, then move beam over off-axis camera using JEOL beam shift knobs.
  5. Uncheck Additional beam shift to store this value.
  6. Take a View picture, and Select Focus under Define Position of Area.
  7. Left-click somewhere on the View screen where you want Focus to be. It will always be along tilt axis.
  8. Select None under Define position of area.
• Set up SerialEM Search mode as follows:
  1. Take a View picture.
  2. Click "search" under Area to show when screen down.
  3. Lower screen.
  4. Set spot size to 5, set mag to 10k, expand beam.
  5. Check then uncheck Continuous update of mag and beam to save settings
     • NOTE JEOL version does not remember diffraction mode as of version 2.74.
  6. Change JEOL to SA DIFF mode.
  7. Move FLC panel CL1 all the way to the right.
  8. Move beam over off-axis camera using PLA shift.
  9. Raise screen.
  10. Move toggle to TEM (from CCD).
  11. Click Unblank Beam on SerialEM low dose control panel. You should see an image on live camera.
  12. Use PLA shift to finely adjust position of live image to match the picture you took in View.

Instructions for using this mode

• To go to Search, make sure that Search radio button is selected under Area to show when screen down.
• Be sure that BLANK beam when screen down is selected.
• Drag FLC Panel CL1 all the way to the right.
• Lower screen, press SA DIFF, raise screen, move toggle to TEM, and click Unblank beam
• You should now see a live image.
• Before taking any View or exposure pictures, be sure to:
  1. Move toggle back to CCD.
  2. Click "All Off" on FLC control panel.
• To go to a live Focus view, instructions same as for search except no fiddling with FLC panel (keep off) or SA DIFF mode.

Visit the Cryo EM Website at http://www.nysbc.org/facilities/CEM

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• Set ALLOWTOPICVIEW =

-- RubenDiaz - 31 Mar 2009