EM Image Processing and Data Analysis


Overall Workflow

In general, workflow looks like this:

  1. Set up your account for TACC and 3dem.org.
  2. Acquire a stack of serial tSEM images (a complete set of original images with optimized focus, brightness, and contrast; Don't forget to take an image of a calibration grid!)
  3. Align the images with AlignEM-SWiFT or Fiji/TrakEM2 on 3dem.org
  4. Stack-crop the aligned images, export as .tif files
  5. Assign a series code and rename image files
  6. Get a spreadsheet ready for series data collection
  7. Import aligned images into PyReconstruct
  8. Manually fine-tune alignment, if necessary
  9. Calibration: x-y (pixel size) with a calibration grid image; z (section thickness) by the cylindrical mitochondria method
  10. Choose and trace neuropil elements (and organelles) of interest
  11. Data analysis
  12. Publication, etc.

See /wiki/spaces/khlab/pages/53543099for what workflow might look like for the KH lab.


Things to do before Reconstructing

Learn about neuropil ultrastructure

!IMPORTANT!  How familiar are you with EM images? Can you identify neuropil, synapses, organelles, etc.?

Get your TACC account

!IMPORTANT!  Make sure you have access to the 3dem.org portal at TACC (James Carson is the current contact person).

Pre-processing of serial section EM images before importing into RECONSTRUCT


Protocols for PyReconstruct