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Contents
User Instructions for 2100F
- Assumed that the microscope has already been aligned and software set up for imaging
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- Some familiarity assumed, as these instructions are not nearly enough for a first-time user
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- All users REQUIRE training with staff before using K2 camera
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- In general, this microscope will be used only for cryo samples
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| | 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
- TEM/CCD toggle to change shuttering control to CCD
iMDS |
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> | 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
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| | Sequence of events
- Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
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- 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 maximized)
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- 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
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- Go to Focus mode, change DM preset to Focus
- Put specimen in focus (zero contrast, Thon rings maximized)
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- Press Mag 1 to reset defocus to 0
- Adjust defocus to desired level
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- Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
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- In EM Menu 3 software, advance image number to a fresh image
- In EM Menu 3, press "Exposure". Should get a flash on Fast scan camera, and a new image should come up
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- In Leginon software, press camera icon to take and save image
- Go back to Search mode. Change DM setting to Search
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- Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
- Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam
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< | Taking a film image |
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> | Advice and Common Issues |
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- To take a film image, be sure that auto-exposure is unchecked
- Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
- After adjusting focus in Focus mode, lower screen
- Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
- After film image taken, you are brought back to focus mode
- Optionally, can take a CCD image after a film image to verify that region is ok.
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| | 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
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- In EM Menu 3 software, press Control TEM to open up a new window
- In Control TEM window, set X tilt angle to 15 degrees, and press execute
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- 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
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- If you are not eucentric, you will see image shift on the Fast scan camera window
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- Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
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- Set tilt angle to 0, and execute tilt. Should see no (minimal) sample movement
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- Reset tilt angle to 0 using JEOL interface. Should see no (minimal) sample movement
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- Close "control TEM" window
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> | Cannot find Focus beam |
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- 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!)
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- In EM Menu 3 software, click "Off-axis" button to move beam over Fast scan camera
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- If beam disappears when moving focus position, call a staff member to redo beam shift / image shift alignment
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| | 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
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- 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
- If it is really bad, try re-centering Focus using PLA as described above
- At a 2 um radius, you often see the edge of the objective aperture in Focus mode
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< | 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:
- Go to Standard Photoset mode (normally not used at all in cycle)
- Press Low Mag button and lower mag to 150X
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> | 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
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- Center beam with beam shift, adjust brightness and spot size to 5
- Raise screen and put CCD/TEM toggle in CCD mode
- Quit EM Menu 3 if it is running
- Start SerialEM. Should hear a beep. If you get an error, quit and restart.
- Set up Record at binning 2, and 0.1 second exposure
- Take a test record image to be sure exposure is good
- Open a new Navigator window
- Choose to make a full grid montage. Change overlap to 15% and choose to ignore pieces to skip.
- Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
- Once done, choose to make a new map.
- If map doesn't look good, try turning off or on "sloppy montage" option and see which is better. Double click map to reload
- 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
- Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
- Optionally, save navigator file
- Quit SerialEM, go back to mag 1 mode
- Cycle through hysteresis cycle a few times. You will likely need to realign things.
- Restart EM Menu 3
- In Search mode, go to saved positions and search there
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- Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
- TEM/CCD toggle should be in CCD position
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- 3/15/15 -- not yet installed on K2
- Never try to use SerialEM and MDS/iMDS at the same time
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- If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
- Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
- For montages, use at least binning 2
- Never try to use SerialEM and MDS/iMDS at the same time, except as described above
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- Never use SerialEM with the standard tilt holder
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- 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)
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-- BillRice - 28 Sep 2007 |
