jadas

fanaticalpumaΜηχανική

5 Νοε 2013 (πριν από 4 χρόνια και 5 μέρες)

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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”

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 Conway

EPU (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

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
(
I
0
/
I
)

I
0

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.

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