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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. 




 



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 [[Presets for MSI-RCT applications|< Presets for MSI-RCT applications]] 

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