Principles & Protocols

Principles - EM Tomography Sample Prep Considerations:

because life’s not 2 dimensional

1. Fixation
   • Chemical fixation
     • Common fixatives: aldehydes and heavy metal fixatives
   • Cryo fixation
     • Cryoprotected
     • Non - Cryoprotected
2. Embedding, Staining & Sectioning
   • Support matrix
     • Cryo (in vitreous ice)
     • Resin (aka: plastic)
   • Post Staining
     • Not necessary for cryo
     • Resin sections: Uranyl Acetate & Lead Citrate
   • Sectioning & section thickness (“resolution” vs. depth in Z)
3. Imaging
   • Dose
   • Tilt angle & # of tilt axes (wedge vs. pyramid)
   • Film or CCD
4. Image Alignment & Reconstruction
   • Alignment
     • Fiducial markers (i.e. colloidal gold)- Least Squares Method
     • Fiducial less alignment
       • Cross-correlation
       • Common line
       • Area Matching
   • Back projection
     • r* weighted back projection
     • SIRT
     • ART
5. Segmentation & analysis (why tracing in kindergarten was so important)

• Set ALLOWTOPICCHANGE = CemfacGroup

-- KakoliMitra - 11 Feb 2009