IntroductiontoAutomatedDatacollection

DavidBushnell,January2019

SLAC

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Agenda

1. Overview of Software Packages for Automated collection S2C2 will be using

2. Workflow for Automated collection3. Common Questions/Problems

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EPU from ThermoFisher Scientific

1. Installation/Setup done by Thermo apps2. Easy to use, walks you through the workflow3. Good integration with Phase Plate4. Good documentation and online training (https://em-

learning.com/)5. Not very flexible if you need non-standard data

collections6. Only Available for Thermo Scientific and FEI systems

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EPU from ThermoFisher Scientific

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SerialEM from University of Colorado Boulder

1. Inexpensive2. More complicated to set up than EPU3. Has a scripting language making it very flexible4. Useful for Single Particle and Tomography5. Steep Learning Curve6. Works on a wide array of TEMs and cameras

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SerialEM from University of Colorado Boulder

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Basic Work Flow

1. Map the Grid (Atlas)2. Choose Squares of Interest3. Choose Holes4. Setup Low Dose5. Run Collection

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Map the Grid (Atlas)

1. Purpose is to get a low magnification overview of your grid

2. Take many images an stich together as a single montage

3. Done at Lowest magnification where fixed apertures do not obstruct your view

4. Make sure to remove objective aperture

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Map the Grid (Atlas)

1. Full Grid Montage using LM 80X and Ceta Camera

2. 6x6 images with ~10% overlap

3. ~15 minutes total acquisition time

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Choosing Grid Square for a Closer look

1. Look for Intact squares

2. Squares that have Ice

3. Take Higher Mag images and determine Z height of the Squares

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Choosing Grid Square for a Closer look

1. Square Maps Acquired at LM 80x on K3

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A Closer Look to Judge the Ice

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Selecting Holes for collection

1. Choose region for collection using a polygon (green lines)

2. Avoid major contamination

3. I don’t recommend selecting the holes adjacent to the grid bars

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Selecting Holes for collection

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Adding Holes for collection (SerialEM)

1. Choose 5 holes.

1. Corner2. Adjacent

Hole N and E3. As N and E

far the line of holes as you can

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Adding Holes for collection

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Adding Holes for collection (SerialEM)

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Adding Holes for collection

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What is Low Dose?

1. Low Dose is the data collection regime used in for dose sensitive samples

2. Objective is to minimize e-3. Three main States, View, Focus and Record

1. View is used to align your target/hole (no stage is perfect)2. Focus is used to set/reset your defocus for collection,

image shifted from Record3. Record is where the magic happens

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Low Dose: View Mode

1. Choose a Mag where you can see several holes (ex Lowest SA 1,700K on Titan Krios)

2. Camera settings and Defocus offset to produce high contrast image (-80 to -100 um works well)

3. Align View to Square maps using shift to marker

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Low Dose: View Mode save a template hole

1. Choose a clean hole2. Adjust the size of the view image

to include the hole and just a little of the surrounding holes

3. Save into image buffer for use in template matching while collecting

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Low Dose: Align View mag to Square map

1. Choose a feature you can see in your Square maps

2. Find that feature in you view mag3. Determine X,Y offset between the

mags

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Low Dose: Align View mag to Square mag

Squaremap ViewMode

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Low Dose: Record Mode (Choosing Magnification)

1. Choose mag based on data collection goal.1. Depending on the detector resolution limit will be ~3-4x the

magnified pixel size2. Do you Need lots of particles to sort out

sample/conformational heterogeneity? Choose a lower mag to increase the imaging area and number of particle per image (~2-3 ang/pixel)

3. Pushing resolution? Choose a higher mag (~0.5-1 ang/pixel)

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Low Dose: Record Mode (Setting Beam conditions)

1. Set your total dose rate and total dose for record1. Adjust spot size, C2 aperture2. On Systems with C3 can adjust beam diameter within

parallel range2. Decide on Defocus range for collection

1. Higher defocus easier to see, limits resolution2. Recommend ~1um range

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Low Dose: Record Mode (Multiple records per Hole)

1. Can I record multiple images per hole?1. How big is your Record beam?

1. Need to make sure the Record/Focus beam does not overlap with the record area (just the detector)

2. Larger holes allow for more images per hole3. Allows for faster data collection using image shift (fast) within

a hole

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Low Dose: Record Mode

1. Example from EPU2. Record areas in Green,

Focus in Blue, Drift test in Purple

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Low Dose: View mag to Record Mag

1. Choose a feature you can see in View mag and record mag

2. Apply Image Shift to View mag so the feature is in the same position in Record and View

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Low Dose: Record Mode align View mag

View Mag Record Mag

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Low Dose: Record Mode align View mag

View Mag Record Mag

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Low Dose: Focus State

1. Use the same mag as record or higher

2. Determine shift along tilt axis where the Focus spot will be over carbon and not impacting your record images

3. Frequency of focusing

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EPU from ThermoFisher Scientific

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Adding Holes for collection

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Basic Work Flow

1. Map the Grid (Atlas)2. Choose Squares of Interest

1. Judge ice2. Determine Z height

3. Choose Holes4. Setup Low Dose

1. Align Mags/Modes2. Determine record mag, Dose and defocus range 3. Set record template and focus spot

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Common Question: How many Squares do I need?

1. How many Grid Squares with Good ice do you need for a data collection

2. ~60 stage shifts an hour when not using image shift (one shot per hole)

3. ~30-40 stage shifts per hour when using image shift (two+ shots per hole)

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Common Question: How many Squares do I need?

Mesh R2/1 R2/2 R2/4 R1.2/1.3 R3.5/1 R1/4 R5/20 R0.6/1

200 900 506 225 1296 400 324 13 3164

300 324 182 81 467 144 117 5 1139

400 160 90 40 231 71 58 2 564

1. ApproximatenumberofHolespergridsquareoncommonQunatifoils

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Common Problems

1. The targeting is far/off doing a bad job1. Check Z height. 2. Ice very thick, or View image needs more contrast (higher

defocus/higher counts)3. Grid is bent4. Template for matching is poor

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Common Problems

1. Drift is really high1. Increase delay after stage motion2. Carbon in the square is broken3. Poor clipping of the grid4. Environmental issues (temperature, dewar bubbling, …)

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Online Resources

1. EPU1. EM Learning (https://em-learning.com/)

2. SerialEM1. Home page (http://bio3d.colorado.edu/SerialEM/)2. YouTube Channel

(https://www.youtube.com/user/BL3DEMC)3. SerialEM google group 4. SerialEM Script Repository

(https://serialemscripts.nexperion.net/)