Leginon

h1. RCT run protocol from a user

h2. Setup

h3. Days before

* Pump down stage

*   Cryo-cycle for 4 hours (240 min)

*   Prepare grids 

h3. 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 

h3. 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|

h3. 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 

h2. 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 

h2. 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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[[RCT calibration| < RCT application calibration]]