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> | General guidelines for leginon use
- Expect to spend the morning getting leginon set up
- You should commit to a possible all-night session if grid is good
- no sample -- basic alignment checks / verification Staff or expert users
- Carbon sample -- focus checks, astigmatism, coma-free Staff or expert users
- Load Cryo sample Staff or advanced users
- Grid image to find square; initial check Staff or experienced users
- Z-focus to make eucentric Staff or experienced users
- Grid atlas Staff or experienced users
- Once grid is good
- pick squares (1 acquire per square)
- best pick one square first, then just a few holes on this square. If good, go back to pick more squares
- Pick good holes
- maybe best to turn off hole finding routine at first
- Pick many holes per square, but only one focus position (used for z-height)
- This step takes awhile
- Re-pick holes for centering step
- one acquire point, does not need to be in exact middle
- no focus point (would be ignored anyway)
- This step takes ~30 min for a "full" square
- Finally pick acquire point
- one point per hole due to beam size
- add one focus point on carbon for drift monitor / focus
Further Notes on j2100
- Add a melt step for focus (20 sec exposure)
- Need to turn on FLC for "gr" preset
- Load "spot 6 for gr" saved FLC preset
- Turn off FLC when done gr atlas
- however, sq is still OK with FLC on. Perhaps leave it on for all Low Mag work?
- preset alignments seem pretty constant. Only check/change if problems seen
- Imaging of ~30 holes took 1.5 h (starting from submitting exposure queue)
- Users strongly encouraged to only use leginon interface unless they are experts
- Use timer for gun valve on exposure step to be safe
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| | iMDS
New Instructions for K2 camera
- Please read and be familiar with use of Leginon and K2 on EMG website
- Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
- Dose (brightness) on all modes should be 10 pA/cm2
- 10.4 pA/cm2 = 8 e/pixel/second
- IMPORTANT: Beam MUST be blanked (live mode off) during mode changes, otherwise camera may be hit by bright beam
- Can run Leginon "Manual" app for movie collection
- Set Camera Mode to Super resolution
- Choose to record movies (frame numbers are irrelevant for K2; all are saved)
- Choose total exposure time and frame exposure time as apprporate
- Be sure that option to save images is checked (under Advanced Settings)
- Makes files smaller (raw images saved as bytes, gain correction and alignment done via Appion)
Good settings
- Search Mode
- alpha 3 (75C0)
- 2000X mag
- To make beam dim enough, change 'Custom" to on and spot size FFFF
- Focus
- alpha 2 (95D0)
- 80,000X mag
- Spot 5
- Beam condensed to just cover camera (only viewing center 1/2 or 1/4)
- Photoset
- alpha 2 (95D0)
- 25,000X mag (~1.4 A/pixel)
- Spot 3-5 (beam very condensed to just cover camera)
- Use DM for live search and exposure
- Search: Full CCD, 4X binning
- Focus: Center 1/2 or 1/4, 1X binning
- Use Leginon for exposure
- images to database
- 3/13/15 -- still saved on local disk, need to be manually transferred to bnc14
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 live view in DM (K2 camera) -- Use Search setting
- Find and center area of interest (use PLA to align Search and Photoset)
- Stop live viewing
- Go to Focus mode, change DM preset to Focus
- Put specimen in focus (zero contrast, Thon rings maximized)
- Press Mag 1 to reset defocus to 0
- Adjust defocus to desired level
- Stop live viewing
- Go to MDS Photo Mode
- In Leginon software, press camera icon to take and save image
- Go back to Search mode. Change DM setting to Search
Advice and Common Issues
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
- In JEOL Oprate window, tilt up in 5 degree increments
- If you are not eucentric, you will see image shift on the live camera window
- Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
- Reset tilt angle to 0 using JEOL interface. Should see no (minimal) sample movement
Cannot find Focus beam
- 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:
- lower screen to see beam
- Make sure PLA button is lit
- Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
- If beam disappears when moving focus position, call a staff member to redo beam shift / image shift alignment
Search and MDS Photo no longer aligned
- Search and Photo may go out of alignment, especially if you leave the hysteresis cycle
- Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
- In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
- Go to Focus, then MDS photoset
- Lower screen
- Use specimen movement to center the object manually
- Go back to Search mode
- Be sure that PLA button is lit
- Use DEF/Stig X and Y to re-center object
Astigmatism in Focus mode
* Try re-centering Focus using PLA as described above
* Do not worry about astigmatism in focus mode. Because of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
- Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
- When happy, write down Objective Stig lens values (in JEOL menu)
- Stop live mode, adjust brightness and mag to original values
- go to Search then Focus
- In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
Apertures visible in Focus and Search
- With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
- Can adjust bright tilt to make beam fully visible
- Note that each mode has its own bright tilt, so this will not affect exposure mode
- good idea to do a few hsyteresis cycles after changing this
Use STD Photoset to getr low-mag views of grid
- Go to Standard Photoset mode (normally not used at all in cycle)
- Press Low Mag button and lower mag to 150X or lower
- manually find and center good squares.
- Store positions in JEOL stage interface for later recall
- Go back to MDS Photoset when done. Leave for 15 minutes to allow lenses to warm back up
- 3/15/15 -- not yet installed on K2
- Never try to use SerialEM and MDS/iMDS at the same time
- Never use SerialEM with the standard tilt holder
-- BillRice - 28 Sep 2007 |
