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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”. - PowerPoint PPT Presentation
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Automation in Single-Particle Electron MicroscopyJian GuanHafenstein 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
Website of National Center for Macromolecular Imaging (NCMI)
Automation
Huge number of Particles Demands
Software systems with various degrees of automation and robustness
• AutoEM semi-automated FEI Tecnai series EM
• Leginon advanced automation Philips CM series and FEI Tecnai
• Serial EM automation tomography FEI and JEOL
• TOM Toolbox FEI
• Batch Tomography for cryo-tomography FEI
• JADAS JEOL
James ConwayEPU (FEI version of Leginon)
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
EPUStands for “E pluribus Unum”, Latin phrase for “out of many, one”
FEI version of LeginonUsing Python programming languageCompatible with both Linux and Windows OS
Work flow
JADAS
Grid Searching
JADAS
100-150X
Grid SearchingLeginon
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
Ulrike Ziese, Dept. Molecular Cell Biology
Autofocus: beam tilt induced image shift
objective lens
specimen
object plane
objective aperture
image plane
defocus
image shift
optical axis
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.
ExamplesJADAS EPU
Speed of data collection
JADAS Using JEM3200FSC, record 30-40 4k×4k CCD images per hour, if each image has 100 particlesOne day: 960 images 96000 particles
EPU
one hour to set up a run for days or weeks,one million particles in a four-daysession on an FEI Titan Krios™ microscope.One day: 250000 particles