EM Image Processing and Data Analysis

Overall Workflow

In general, workflow looks like this:

Set up your account for TACC and 3dem.org.
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!)
Align the images with AlignEM-SWiFT or Fiji/TrakEM2 on 3dem.org
Stack-crop the aligned images, export as .tif files
Assign a series code and rename image files
Get a spreadsheet ready for series data collection
Import aligned images into PyReconstruct
Manually fine-tune alignment, if necessary
Calibration: x-y (pixel size) with a calibration grid image; z (section thickness) by the cylindrical mitochondria method
Choose and trace neuropil elements (and organelles) of interest
Data analysis
Publication, etc.

See here for what workflow might look like for the KH lab.

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

Atlas of Ultrastructural Neurocytology and Tutorials
Peters A, Palay SL, Webster H. (1991) The Fine Structure of the Nervous System (3rd Ed). Oxford University Press, New York. ISBN 0-19-506571-9.
- For KH lab – we have this book on our file server.
Electron Micrographs of the Neuropil: Basic Literacy by Patrick Parker

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

Upload serial section EM images to 3dem.org
- Command line instructions (Harris lab specific; restricted access)
Alignment of serial section EM images using AlignEM-SWiFT
- old protocol for aligning serial section EM images using Fiji /TrakEM2
Assign a series code (Harris lab specific; restricted access)
Rename aligned serial EM image files with Bulk Rename Utility on Windows PC, or use Bulk Rename software if you are on stampede2 at TACC.
- Suggested format: XXXXX_### (XXXXX = series code; ### = section number; 000 = calibration grid image)