Processing

FIB-SEM data

Make a stack of images

1. Copy data over from microscope
2. Select useful images
3. Make an mrc stack from several tif images using IMOD

   •  > tif2mrc Image*.tif rawstack.mrc 

Correct for gradient/shading in images

1. To work on entire stack convert mrc file to tif file

   •  mrc2tif -s rawstack.mrc rawstack.tif 

2. Open tif stack in ImageJ^?
   • may need more memory than standard ImageJ^?
   • "which ImageJ^?" to find where the program is then "more ImageJ^?" and increase memory (/cryoem/ImageJ)
3. To correct the shading: Plugins> Filters>Fit polynomial
4. Adjust Brightness/Contrast to whole stack
5. Save as new tiff stack

Decurtain stack

Curtain Removal

• Curtaining seems to be caused by surface damage, or milling a rough surface
• Best way is to prevent them from happening at all by minimizing viewing with ion-beam, particularly at high current (>80 pA)
• Also pre-coat with Pt using e-beam, then coat with ion beam at minimum reasonable current (>10 min per area)

To remove using ImageJ^?:

• Manually design a filter
  • Do Process: FFT of a sample image in stack
  • In FFT, box out small rectangles along middle of image
    • Example: FFT is 2048x2048: box out x-sixe=374, y-size= 14; xcenter = 1116 or 578; ycenter=1016
    • Need to box both sides
  • For each box, choose to Fill with black
  • Do inverse FFT
  • Should see reduction in curtains. Will need to play with size and positioning to optimize curtain removal and minimize artifacts
• Make the filter
  • Make a new blank image: 8 bit; white background. Size should be 2Kx2K (all dimensions <2K pixels)or 4K x 4K (if any dimension > 2K pixels)
  • Mark out same boxes as above. If scale doubled, adjust accordingly
  • Save as Filter.tif * sample decurtaining filter:
    
• Apply Filter:
  • Choosr Proxess -> FFT --> Custom filter
  • Choose Filter.tif as input
  • BUG: Choose to process entire stack. Only one image is processed. Then choose again, deselect entire stack. Entire stack will be processed
• NOTE: top and bottom edges will not be filtered, so it is best to do this on an uncropped image
• If ImageJ^? won't open stack:
  • Open raw tifs as image sequence in ImageJ^? and save as a new image sequence
  • Convert filtered tifs to stack with tif2mrc
• AMIRA
  • May have to process partial stacks due to memory issues
  • Image Processing/Frequency Domain/FFT Filter
  • Use default settings

Etomo

Align Serial Sections

• Choose stack
  • Single Frame
• Align: Initial Auto Alignment (cannot use warping alignment files)
  • Apply Sobel Filter
  • Search For: Rotation/Translation
  • Binning in search: 1
• Align: Refine with Auto Alignment
  • Find warping transformations: (can repeat warping process as needed)
    • Correlation patch size (1024, 512)
    • Use boundary model: create one by selecting area of interest across stack, must manually select "model" mode
    • Limits to shifts in x and y (5, 10)
• Make Stack: Make Aligned Stack
  • Remove trends in translation & rotation
  • Enter size in x and y (adjust for shift obtained from initial alignment file)
  • Enter shift in x and y

IMOD

Crop volume

• Use rubberband tool to define area
• Shift + I pops up trimvol command
  • Copy and paste code, adjust the z value to include all sections, and add the input and output stacks:

     >  trimvol -x 295,3161 -y 1670,3642 -z 1,300 alstack.mrc alstack_crop.mrc 

Manual Processing

Non-Orthogonal Viewing

• For data collected at a non-orthogonal viewing angle (e.g. 38 degrees), use -n 0 option to eliminate sample drift afterwards, view the beginning and the end of the file to determine how much padding will need to be added in y to allow for drift

 >  xftoxg -n 0 rawstack.xf rawstack.xg

Use newstack to apply these alignments, with final size adjustment due to shifting of images Example: if top image has shift of -400, and bottom image has shift +400, need to adjust y-size by 800. 3536 + 800 = 4336

 >  newstack -size 4096,4336 -xform rawstack.xg rawstack.mrc alstack.mrc

• Use the warped .xg for transforms NOT the pre-warp .xg

• to obtain SEM info:

---++Movie making - http://www.nysbc.net/twiki/bin/view/Main/MovieMaking

1. Open stack in IMOD
2. File > Movie/Montage
   • Looping - One way
   • Snapshot - JPEG
3. Make zap files - keep movie window open and play in zap window to save zap images
4. Put zap???.jpg into a separate folder

5. jpeg2yuv -f 24 -I p -L 0 -j zap%03d.jpg | yuv2lav -f a -o movie.avi

6. ffmpeg -i movie_48fps.avi -vcodec mpeg4 -b 4800k movie_48fps.mp4

---++Join Serial Tomograms

1. Make merge directory and create soft links to stacks

   ln -s ../031815/alstack1.mrc

2. Open Etomo
   • "Join Serial Tomograms"
   • Choose working directory and output file name
   • Add stacks in order and choose 5-6 slices at start and end to match up (remember the top and bottom are reversed!)
   • "Make Samples"
   • "Align", rotation/translation, "Initial Auto Alignment"
   • "Midas" (optional) "Refine" (optional)
   • "Join", "Get Max Size and Shift", "Trial Join" with every 2 slices, binning 2 and open in 3dmod
   • "Finish Join"
   • Be sure to rename .join output file to .mrc!

• Set ALLOWTOPICVIEW =

-- AshleighRaczkowski - 10 Feb 2015

* movie_48fps.mp4: test movie