RCT run protocol from a user¶

Setup¶

Days before¶

• Pump down stage
• Cryo-cycle for 4 hours (240 min)
• Prepare grids

Scope alignments¶

2.2.1 no sample

gun shift/tilt
    condenser aperture
    condenser stig.
    check if cold trap is in
    adjust eye pieces

2.2.2 with sample

z-height
    focus both M and SA modes
    objective stig.
    objective aperture at hi mag, use focus to shrink beam, not intensity
    at zero defocus
        beam tilt pivot points X,Y
        rotation center

Presets and Magnifications¶

preset	mag	camera config	defocus	notes
gr	120X	512x8	0	do not cycle through
sq	1700X	1024x4	-3.0e-4	for square_centering
cs	1700X	1024x4	-3.0e-4	for rct_targeting, copy of sq
hl	5000X	512x8	-5.0e-5	image shift alignment and z-focus
en	50kX-100kX	4096x1	-1.5e-6 to -3.5e-6
fa	50kX-100kX	512x4	-1.5e-6	beam focus and drift checks

Leginon¶

2.4.1 pre-Altas

beam shift/intensity for each preset
    align image shifts btw presets
    matrix calibrate, stage position for gr preset
    optional: fine tune rotation center in Leginon, available in any focus node using the fa preset
    on rare occasions: check optical axis using Leginon tool, this is only required if the square is shifting alot when you tilt the stage

2.4.2 post-Atlas

matrix calibrate, stage position for sq preset
        error ~50%
        shift ~25%
        num position ~2-3
        interval ~1-3e-6 
    RCT node calibration
        make sure the number of features is between 700 and 1500
        take a picture both with and without the grid bar
        take a picture both untilted and tilted 
    calibrate dose
    RCT node setup
    re-align presets
    re-center objective aperture

Running Leginon RCT¶

Start a square:
        Square Targeting (if you have an atlas)
            Go to "Square Targeting" 
            Hit the reload button
            Select a new square with the green cross
            Click the play |> button 
        Simulate Square (if you do NOT have an atlas)
            Go to "Square" 
            Hit the simulate target button 
    Square centering
        When Leginon is finished, a question mark will appear next to the "Square Centering" node
        Click on the "Square Centering" node
        Select one z-focus position (blue cross) and one imaging position (green position) close to the center of the square
        Click the play |> button 
    RCT Targeting
        When Leginon is finished setting the z-height, a question mark will appear next to the "RCT Targeting" node
        Click on the "RCT Targeting" node
        Now you select all the holes to image (not too close to the edge) and a single focus position
        Optional: obtain a picture of the tilted square
            Click on the "Navigation" node
            Open up VNC viewer of the tecnai
            In the VNC window, set the tilt angle to the desire angle
            In the "Navigation" node, collect an image by clicking on the acquire button (camera logo) at the top
            Important, in the VNC window, return the tilt angle to zero degrees, otherwise all your images will fail
            Now select you targets in the "RCT Targeting" node and view the tilted image in the "Navigation" node. Jump back and forth between the two nodes until you select all holes present in both images. 
        When finished, hit the play |> button 
    Focusing
        If manual focusing is enabled, wait for the manual focus window to pop up
        focus the microscope, reset the defocus, and click the red stop button. 
    Watch your images in the "RCT" node

Nitrogen fill¶

Since RCT only focuses once per square, when you fill the dewar, you must refocus to account for changes in the z-height and focus during refill and there is no check for drift calm down so you must manually wait for drift to subside.

Steps in Nitrogen fill:

Pause RCT node
    Wait until Leginon is paused
    Fill holder and cold trap dewars
    Turn off lights
    Check if objective aperture is inserted
    RESET TIMER
    Focus
        simulate z-focus
        send hole preset to scope
        navigate to good focus spot in navigator
        simulate focus in the focus node
        repeat focus until drift subsides 
    If there is an empty hole: you can measure dose or collect bright fields
    Declare drift
    Hit play button on RCT node
    Check images to make sure they are good
    Uncheck user verification of targets

This page was last modified 16:45, 16 July 2010.

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