lecture9

Report
Automation in Single-Particle
Electron Microscopy
Jian Guan
Hafenstein Lab
The definition of Automation
“automation is a comprehensive and versatile strategy that
can deliver biological information on an unprecedented scale
beyond the scope available with classical manual approaches”
Automation in Single-Particle Electron Microscopy: Connecting the
Pieces, Lyumkis D, et al. Methods in Enzymology, Volume 483,2010
Huge
number of
Particles
Demands
Website of National Center for Macromolecular Imaging (NCMI)
Automation
Software systems with various degrees of automation and
robustness
• AutoEM
semi-automated
• Leginon
advanced automation Philips CM series and FEI Tecnai
• Serial EM
automation tomography FEI and JEOL
• TOM Toolbox
FEI Tecnai series EM
FEI
• Batch Tomography for cryo-tomography FEI
• JADAS
James Conway
EPU (FEI version
of Leginon)
JEOL
Susan Hafensein
Outline
•
•
•
•
General Information
Work flow
Grid Searching
Selective squares on the grid
according to ice thickness
• Centering the holes
• Focus and Astigmatism Correction
• Modularity and Flexibility of JADAS
• Examples
• Speed of data collection
General Information
JADAS
JEOL Automated Data Acquisition System (JADAS) software was
developed by JEOL collaboration with NCMI Baylor college of
Medicine
Developed for current generation of JEOL instruments.
Written with C# programming language.
Runs only on Windows OS with at least 1GB memory
EPU
Stands for “E pluribus Unum”, Latin phrase for “out of many, one”
FEI version of Leginon
Using Python programming language
Compatible with both Linux and Windows OS
Work flow
JADAS
Grid Searching
JADAS
100-150X
Grid Searching
Leginon
165X-320X
500-1000 squares
Selective squares on the grid according to ice thickness
The thickness of a vitreous ice layer can be estimated as
(Eusemann et al., 1982; Lepault et al., 1982)
t < K ln(I0/I)
I0 is the intensity of a bright-field image in the absence of ice and I is the
intensity of the image in the presence of an ice layer of thickness t. K is a
constant that is dependent on the geometry of the microscope.
Thickness Range: 500 to 1500 Å
Intensity Based Hole selection GUI automatic local search for JADAS
Ice filter for EPU (Leginon)
Centering the holes
Jadas
Focus and Astigmatism Correction
Autofocus: beam tilt induced image shift
optical axis
specimen
defocus
object plane
image shift
objective lens
objective aperture
image plane
Ulrike Ziese, Dept. Molecular Cell Biology
Ligenon and JADAS can analyze a diffractogram for stigmatism
correction also. When the specimen is on a holey carbon film, the
software can automatically adjust the objective lens stigmator by
measuring the ellipticity of the contrast transfer function rings to
correct the astigmatism.
Modularity and Flexibility of JADAS
• Coupling with other tools developed elsewhere.
EMEN2
• integrate image processing function from
EMAN to assess the data quality in real time
• Off-the-shelf remote logon software (e.g.
WebEX: http://www.webex.com/) to monitor
the data
• collection process and even operate JADAS
remotely.
Examples
JADAS
EPU
Speed of data collection
JADAS
Using JEM3200FSC, record 30-40 4k×4k CCD
images per hour, if each image has 100 particles
One day: 960 images
96000 particles
EPU
one hour to set up a run for days or weeks,
one million particles in a four-day
session on an FEI Titan Krios™ microscope.
One day:
250000 particles

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