data collection (serialem) - stanford-slac cryo-electron … · 2019-09-11 · •data collection...

Data Collection (SerialEM)Corey Hecksel

9/13/19

Requirements of Data Collection Software?

• Image Viewer• Must communicate with camera

software

• Low Dose System • View, Focus/Track, Record

•Navigator System

•Optional: Scripting

Data Collection Strategy?

Tomography Single Particle

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…

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

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

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

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

Introduction to Navigator

•Used to navigate the grid, save positions, correlate images collected at different mags

• Set up batch positions

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

Grid Square Selection

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”

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

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

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.

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

Setting Up Low Dose: View ModeSquare map View Mode

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?

Setting Up Low Dose: View and Record Mode

Record Mag View Mag

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”

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

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

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

What about Tomography?

What about Tomography?

•Requires an anchor map to accurately recall the exact position

•RealignToItem allows features to be accurately recalled

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

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

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/