Difference: CemProtocET (9 vs. 10)

Revision 1021 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
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Principles

EM Tomography Sample Prep Considerations:

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>		Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM
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Principles & Protocols

Principles - EM Tomography Sample Prep Considerations:

 

because life’s not 2 dimensional

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Jump to Protocol

 
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  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)
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Protocols

 
Public website mirror

-- KakoliMitra - 11 Feb 2009

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