Difference: CemProtocET (1 vs. 11)

Revision 1126 Oct 2009 - Main.KdDerr

META TOPICPARENT	name="CemProtoc"

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

Principles & Protocols

Principles - EM Tomography Sample Prep Considerations:

because life’s not 2 dimensional

Changed:

<
<

Jump to Protocol

>
>

Jump to Protocol

Fixation
- Chemical fixation
  - Common fixatives: aldehydes and heavy metal fixatives
- Cryo fixation
  - Cryoprotected
  - Non - Cryoprotected
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)
Imaging
- Dose
- Tilt angle & # of tilt axes (wedge vs. pyramid)
- Film or CCD
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
Segmentation & analysis (why tracing in kindergarten was so important)

Public website mirror

Set ALLOWTOPICCHANGE = CemfacGroup

-- KakoliMitra - 11 Feb 2009

Revision 1021 Sep 2009 - Main.KdDerr

   META TOPICPARENT   name="CemProtoc"
-  META TOPICPARENT
+ name="CemProtoc"
-<
<
+ Principles 
 EM Tomography Sample Prep Considerations:
->
>
+Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM
->
>
+ Principles & Protocols 


 Principles - EM Tomography Sample Prep Considerations:
  because life’s not 2 dimensional
->
>
+ Jump to Protocol
-<
<
  Fixation 
 Chemical fixation  
 Common fixatives: aldehydes and heavy metal fixatives
 
  Cryo fixation 
 Cryoprotected
  Non - Cryoprotected
 
 
  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)
 
  Imaging 
 Dose 
  Tilt angle & # of tilt axes (wedge vs. pyramid)
  Film or CCD
 
  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
 
 
  Segmentation & analysis (why tracing in kindergarten was so important)
-<
<
+ Protocols
 Public website mirror

 

 Set ALLOWTOPICCHANGE = CemfacGroup
 

-- KakoliMitra - 11 Feb 2009

Revision 926 Aug 2009 - Main.KdDerr

META TOPICPARENT	name="CemProtoc"

Principles

Added:

>
>

EM Tomography Sample Prep Considerations:

because life’s not 2 dimensional

Fixation
- Chemical fixation
  - Common fixatives: aldehydes and heavy metal fixatives
- Cryo fixation
  - Cryoprotected
  - Non - Cryoprotected
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)
Imaging
- Dose
- Tilt angle & # of tilt axes (wedge vs. pyramid)
- Film or CCD
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
Segmentation & analysis (why tracing in kindergarten was so important)

Protocols

Public website mirror

Set ALLOWTOPICCHANGE = CemfacGroup

-- KakoliMitra - 11 Feb 2009

Revision 810 Aug 2009 - Main.BillRice

META TOPICPARENT	name="CemProtoc"

Principles

Changed:

<
<

Protocols

>
>

Protocols

Public website mirror

Set ALLOWTOPICCHANGE = CemfacGroup

-- KakoliMitra - 11 Feb 2009

Revision 710 Aug 2009 - Main.BillRice

META TOPICPARENT	name="CemProtoc"

Changed:

<
<

>
>

Principles

Deleted:

<
<

NYSBC|Cryo-EM P&P: Electron tomography

Changed:

<
<

>
>

Protocols

Deleted:

<
<

Principles & Protocols

Interested in EM?	Using CEMfac^?	Principles & Protocols	CEMfac Equipment	Seminars & Courses
CEMfac Home^?	CEM Introduction	Microscope Schedule	NYSBC Directory	Cryo-EM public website

Electron tomography

Principles
Background

Protocols
Protocols

Principles
Background
Text

Protocols
Protocols
Text

Public website mirror

Set ALLOWTOPICCHANGE = CemfacGroup

-- KakoliMitra - 11 Feb 2009

Revision 612 Feb 2009 - Main.KakoliMitra

Changed:

<
<

Revision 5 is unreadable

>
>

META TOPICPARENT	name="CemProtoc"

NYSBC|Cryo-EM P&P: Electron tomography

Principles & Protocols

Interested in EM?	Using CEMfac^?	Principles & Protocols	CEMfac Equipment	Seminars & Courses
CEMfac Home^?	CEM Introduction	Microscope Schedule	NYSBC Directory	Cryo-EM public website

Electron tomography

Principles
Background

Protocols
Protocols

Principles
Background
Text

Protocols
Protocols
Text

Public website mirror

Set ALLOWTOPICCHANGE = CemfacGroup

-- KakoliMitra - 11 Feb 2009

Revision 512 Feb 2009 - Main.KakoliMitra

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> >	Revision 5 is unreadable

Revision 412 Feb 2009 - Main.KakoliMitra

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Revision 312 Feb 2009 - Main.KakoliMitra

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Revision 212 Feb 2009 - Main.KakoliMitra

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Revision 111 Feb 2009 - Main.KakoliMitra

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> >	Revision 1 is unreadable

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