automation in single-particle electron microscopy

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