data collection (serialem) - stanford-slac cryo-electron … · 2019-09-11 · •data collection...
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Data Collection (SerialEM)Corey Hecksel
9/13/19
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Requirements of Data Collection Software?
• Image Viewer• Must communicate with camera
software
• Low Dose System • View, Focus/Track, Record
•Navigator System
•Optional: Scripting
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Data Collection Strategy?
Tomography Single Particle
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Data Collection Strategy?
Tomography
•Dose Symmetric
•Grouped Dose Symmetric
•Bi-directional
•Asymmetric Bi-directional
•Continuous rotation?
•And more…
Single Particle
• Image-shift data collection
•Tilted data collection
•Tilt pair
• Focal pairs
•And more…
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Benefits of SerialEM • Highly Adaptable -> Easy to Script
• Free to use (pay for support)
• Available across multiple microscopes -> large user community• Microscope: Thermo Fisher, JEOL, Hitachi• Detectors: Gatan, Thermo Fisher, DirectElectron, TVIPS, AMT, EMSIS, JEOL, STEM• Computer: Windows 2000, Windows XP, Windows 7
• Robust and Quick Bug/Feature Response
• Unified data collection software regardless of data collection strategy
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Work Flow for Single Particle Data Collection
• Camera Settings
• Location of Regions to Image• Creation of Grid Map• Selection of Squares• Selection of Holes
• Data Collection and Monitoring• Low Dose
•Other Useful Features
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Camera Settings
• Camera Selection• Can use multiple cameras
• Imaging Mode• Presets for different modes ->
View/Focus/Record, etc.
• Readout area• What areas of the camera to readout• Binning
• Exposure Time
• Camera Settings• Gain Reference, Continuous vs Single Image,
Shutter Mode, Counting/Linear/Super-Resolution
• Fractions and Saving
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Work Flow for Single Particle Data Collection
• Camera Settings
• Location of Regions to Image• Creation of Grid Map• Selection of Squares• Selection of Holes
• Data Collection and Monitoring• Low Dose
•Other Useful Features
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Introduction to Navigator
•Used to navigate the grid, save positions, correlate images collected at different mags
• Set up batch positions
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Grid Atlas
• Full grid montage: LM 80-150x
• About 15-20 min acquisition time per montage
• Scripts can be used to montage all grids in the autoloader
• Find intact grid squares with good ice -> requires confirmation at higher mags
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Grid Square Selection
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Setting up Grid of Points
•Optional: Use Polygon tool to select area for hole selection
• Step 1: Place 5 points to define the geometry of the holes
• Step 2: Generate grid of points
• Step 3: Set all points to “Acquire”
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Work Flow for Single Particle Data Collection
• Camera Settings
• Location of Regions to Image• Creation of Grid Map• Selection of Squares• Selection of Holes
• Data Collection and Monitoring• Low Dose
•Other Useful Features
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What is Low Dose?
•Data collection strategy used to minimize dose for dose sensitive samples
•Three Main States: View, Focus, and Record• View is used to align your target/hole• Focus is used to set/reset your defocus for collection, image shifted from your
Record area
• Record is used for data is collection
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Setting Up Low Dose•View
• Camera settings and defocus offset to produce high contrast images
• Choose a mag to give you slightly more than a single hole
• Focus/Trail: • Usually same imaging conditions as Record• Set off-set so that Record and and Focus/Trail do
not overlap
•Record:• Any additional beam shifts should be with respect
to record.
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Setting Up Low Dose: View Mode• Save a clean hole as a template for
hole finding• Should have uniform ice• No contamination• Not Empty
•You should align view mag to your grid square images• Find a piece of contamination in the
grid square
• Mark a point• “Align to Item” in view
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Setting Up Low Dose: View ModeSquare map View Mode
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Setting Up Low Dose: Record
•Choose mag based on imaging goals -> balance target resolution and number of particles
• Set image conditions based on total dose for record• For Talos: Adjust spot size and C2• For Krios: Adjust spot size, C2, and beam “intensity”
•Decide on defocus range• Higher defocus limits resolution but easier to see
• Image Shift Data Collection?
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Setting Up Low Dose: View and Record Mode
Record Mag View Mag
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Image Shift Data Collection
•Users can choose between:• Multi-shot per hole• Multiple holes• Combination
•Users define a image shift “template”
•Must make sure to calibrate “image shift vs. coma”
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Data Collection Scripts
1. Moves to the grid hole
2. Acquires view image and aligns to hole template
3. Focus
4. Acquire image
5. Repeat… many times
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Work Flow for Single Particle Data Collection
• Camera Settings
• Location of Regions to Image• Creation of Grid Map• Selection of Squares• Selection of Holes
• Data Collection and Monitoring• Low Dose
•Other Useful Features
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Other Useful Features
• SerialEM can measure and correct for astigmatism by fitting Thon rings in the FFT
•Coma can be visualized using a Zemlin Tableau
• SerialEM contains methods for automated coma free alignment
•Auto eucentricity and focus
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What about Tomography?
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What about Tomography?
•Requires an anchor map to accurately recall the exact position
•RealignToItem allows features to be accurately recalled
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What about Tomography?
•Very flexible -> Many options
•Can change values per tilt-series in batch collection mode
•Data collection times vary based on which parameters are used
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Common Problems
•Targeting is doing a bad job• Check Z height• View mode does not have enough contrast -> Thick Ice, Dose too low, etc. • Grid is bent• Hole template is bad
•Drift is really high• Turbo pump for autoloader is on• Increase delay after stage move• Carbon is broken• Poor clipping• Environmental Issues
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Online Resources
• SerialEM Home Page: http://bio3d.colorado.edu/SerialEM/
•YouTube Channel: https://www.youtube.com/user/BL3DEMC
• SerialEM Google Group
• SerialEM Script Repository: https://serialemscripts.nexperion.net/