Page History

Overall Workflow

1. 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!)
2. Image alignment with Fiji/TrakEM2 or SWiFT-IR
3. Stack-crop the aligned images, export as .tif files
4. Assign a series code and rename image files
5. Get a spreadsheet ready for series data collection
6. Import aligned images into RECONSTRUCT
7. Manually fine-tune alignment, if necessary
8. Calibration: x-y (pixel size) with a calibration grid image; z (section thickness) by the cylindrical mitochondria method
9. Choose and trace neuropil elements (and organelles) of interest
10. Data analysis
11. Publication, etc.

Things to do before Reconstructing:

!IMPORTANT!  Make sure you have access to TACC (John is the current contact person).

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

• Upload serial section EM images to 3DEM.org
• Alignment of serial section EM images using Fiji/TrakEM2:
  • old protocol for TEM images
  • new protocol for tSEM images using TACC environment (coming soon)
• Assign a series code (Harris lab specific)
• Rename aligned serial EM image files with Bulk Rename Utility
  • Suggested format: XXXXX_### (XXXXX = series code; ### = section number; 000 = calibration grid image)

Protocols for Reconstruct (and other analysis tools):

• Get your Reconstruct here! (Use 32-bit version; 64-bit may be buggy)
• Instructions for new users of Reconstruct: Reconstruct: Intro for Beginners, Reconstruct Basics, User Manual.
• Starting a New Series 
• Section Thickness 
• Calibration Error Correction Strategy
  
• Adjusting brightness/contrast
• Editing Trace Pallettes
• Importing Traces
• Unbiased 3D stereology
• Dendrites:
  
  • Counting/tracing mictotubules
  • Tracing Z-Lengths
  • Tracing plinn (linear nearest-neighbor distance between protrusions)
• Spines:
  
  • Stamping Dendritic Protrusions
  • Cutting off spines and spine heads: Reconstruct / Blender (with Python add-on described in Bartol et al., 2016) 
• Synapses:
  
  • Identifying Symmetric and Asymmetric Synapses
  • Tracing synapses (nascent and active zones)
• Axons:
• Organelles:
  • Ribosome Identification
  • SER and Spine Apparatus Identification
• Exporting quantitative data
• RECONSTRUCT color scheme
• pyRECONSTRUCT merge tool
• CellBlender: Github | MCell and CellBlender | Tutorial
• NeuroMorph: http://cvlab.epfl.ch/NeuroMorph

Data Analysis Protocols:

• Data Entry and Dendrite Analysis Spreadsheet
• Statistical Analyses with STATISTICA
• Making Box and Whisker Plots in Excel
• Data Transforms
• Statistics for Biologists (Nature collection)