RCT calibration » History » Revision 3
Revision 2 (Anchi Cheng, 05/26/2011 03:33 PM) → Revision 3/7 (Neil Voss, 08/13/2012 06:02 PM)
h1. RCT application calibration RCT type of data collection need the normal MSI calibration. In addition, the followings are also required. * image shift matrix and modeled-stage calibration (scale and rotation only) at the 1700x preset used for select RCT targets * recently saved rotation center in the database (You can do so in "Beam Tilt" node in "Calibration" application. h1. Dump from other wiki 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 ↓ ccd ↓ 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 -- -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. ______ [[Presets for MSI-RCT applications|< Presets for MSI-RCT applications]] ______