Confidential
Gatan Webinar Series
Automated TEM Tomography AcquisitionFebruary, 2008
Questions?
2
Tomography Webinar Overview
Tomography In Practice
General Tomography Setup
General Microscope Preparation
Microscope Calibrations
TEM Tomography Setup
Tomography specific microscope preparation
TEM Tomography Operation
Startup
Running
Paused
Manual assist
Working With Tomography Data
3
Principles Of Tomography
4
Tomography Data can be used to give a reconstruction the 3D
volume that gave rise to the projections
Tomography In Practice: Microscope
Issues
5
e-
•Each tilt introduces shifts in
X, Y, and Z
•Drifting in X, Y occurs for
short time after a tilt change
Pictured Above: A Gatan 912 Holder for High Tilt Tomography
Tomography In Practice: Computer Issues
Memory limited to approximately 1GB blocks on a 32-bit system
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Integer (2 byte) Float ( 4 byte)
256 x 256 x 128 16 MB 31 MB
512 x 512 x 128 64 MB 128 MB
1024 x 1024 x 128 256 MB 512 MB
2048 x 2048 x 128 1 GB 2 GB
4096 x 4096 x 128 4 GB 8 GB
Tomography Setup Checklist
Align Microscope
Do Z height alignment
Beam tilt pivot points
Rotation center alignment
Calibrations (if necessary)
Magnification Calibration
Setup Tomography Calibrations
Run Tomography Calibrations
Beam Tilt/Image Rotation
Image Shift
Beam Shift
Induced Beam Shift
Find Suitable Specimen Area
Find feature of interest
Confirm Z Height Alignment
Explore full tilt range
Setup Tomography
Setup Camera
Setup Tomography Parameters
Tilt Angles
Tracking
Memory
Remove backlash in goniometer
Start Data Collection
7
Microscope Alignment
Digital Micrograph Help System
Daily Alignments
Insert And Align Condenser Aperture
Gun Shift Alignment (Align spot sizes)
Z Height Alignment
Optional Alignments
Beam Tilt Pivot Points
Voltage Center or Current Center Alignment (Rotation Alignment)
Insert Objective Aperture
Objective Astigmatism
8
Microscope Preparation
9
Help files are installed with all released software
Z Height Adjustment
Sometimes called mechanical eucentric height
NOT optical eucentric height
Tilt the specimen and look for minimum movement
10
e-
Z Height
e-
0° Specimen 45° Specimen
e- e-
Adjust the ratio of the Beam
Shift/Beam Tilt deflector coils so
beam tilt does not introduce a beam
shift
If the specimen is in focus the image
shouldn’t move in response to an
beam tilt
Beam Tilt can give us an accurate
measure defocus.
Beam Tilt Pivot Points
11
Condenser
Lens
Beam Shift and
Beam Tilt
Deflectors
Specimen Area
Rotation Center Alignment
Wobble the Objective Lens (or if the
option is available HT)
Adjust beam tilt until rotation occurs
around center of screen
May have to repeat Beam Tilt Pivot
points alignment
12
Objective
Lens
Specimen
Tomography Setup Checklist
Align Microscope
Do Z height alignment
Beam tilt pivot points
Rotation center alignment
Calibrations (if necessary)
Magnification Calibration
Setup Tomography Calibrations
Run Tomography Calibrations
Beam Tilt/Image Rotation
Image Shift
Beam Shift
Induced Beam Shift
Find Suitable Specimen Area
Find feature of interest
Confirm Z Height Alignment
Explore full tilt range
Setup Tomography
Setup Camera
Setup Tomography Parameters
Tilt Angles
Tracking
Memory
Remove backlash in goniometer
Start Data Collection
13
Tomography In Digital Micrograph
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GMS 1.7.1
Microscope Calibrations
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Microscope Calibrations
Microscope Calibration Help Page
Magnification Calibration
Calibration Setup
Defocus Calibration Factor
Beam Tilt
Image Filtering
TEM Calibrations
Beam Tilt
Image Rotation
Image Shift
Beam Shift
Induced Beam Shift
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Microscope Calibrations
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Magnification Calibration
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Calibration Setup
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Image Filtering
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Microscope Calibrations
Calibration Help Page
Magnification Calibration
Calibration Setup
Defocus Calibration Factor
Beam Tilt
Image Filtering
TEM Calibrations
Beam Tilt
Image Rotation
Image Shift
Beam Shift
Induced Beam Shift
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TEM Calibrations
22
TEM Calibration Dependencies
Global Calibration
Beam Tilt
Calibrations For Every Magnification
Image Shift
Beam Shift
Induced Beam Shift
Calibrations For Every Magnification and Camera
Image Rotation
23
TEM Calibration Dependencies
Changes To Microscope Changed Calibration
Setup
Changed Calibrations Other
Normal
Daily
Alignment
Poor Pivot
Point
Alignment
Poor
Rotation
Center
Alignment
Service
Engineer
Makes
Large
Alignment
Change
Change
Defocus
Calibration
Factor
Change
Applied
Beam Tilt
Value
Change
Image
Filtering
Parameters
Beam Tilt Image
Rotation
Image Shift Beam Shift Induced
Beam Shift
Digital
Micrograph
Upgrade
Beam Tilt
Image
Rotation
Image Shift
Beam Shift
Induced
Beam Shift
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Previous Calibration Invalid. Re-Run Calibration
Calibration Still Valid
Tomography Setup Checklist
Align Microscope
Do Z height alignment
Beam tilt pivot points
Rotation center alignment
Calibrations (if necessary)
Magnification Calibration
Setup Tomography Calibrations
Run Tomography Calibrations
Beam Tilt/Image Rotation
Image Shift
Beam Shift
Induced Beam Shift
Find Suitable Specimen Area
Find feature of interest
Confirm Z Height Alignment
Explore full tilt range
Setup Tomography
Setup Camera
Setup Tomography Parameters
Tilt Angles
Tracking
Memory
Remove backlash in goniometer
Start Data Collection
25
Explore The Full Tilt Range
Tomography Setup Checklist
Align Microscope
Do Z height alignment
Beam tilt pivot points
Rotation center alignment
Calibrations (if necessary)
Magnification Calibration
Setup Tomography Calibrations
Run Tomography Calibrations
Beam Tilt/Image Rotation
Image Shift
Beam Shift
Induced Beam Shift
Find Suitable Specimen Area
Find feature of interest
Confirm Z Height Alignment
Explore full tilt range
Setup Tomography
Setup Camera
Setup Tomography Parameters
Tilt Angles
Tracking
Memory
Remove backlash in goniometer
Start Data Collection
27
Camera Setup
28
Tomography Setup
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Tomography Setup: Tilt Angles
Set up the tilt range you want to
cover during data collection
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Tomography Setup: Tracking
X,Y Tracking
• None
• XY Manual Tracking
• XY Tracking with reference
Image
Z Tracking
• None
• Manual Focusing
• Beam Tilt Induced Shift
AutoFocus
31
Tomography Setup: Memory
Double check that current tilt series
does not exceed computer memory
Can choose how to save data
32
Tomography Setup: General
Save and load previous
tomography parameters
33
Remove Backlash From Gears
Step a few degrees past
the angle you want to start
imaging.
Then move one or two
degrees in the same
direction you will collect
your data.
34
Tomography Setup Checklist
Align Microscope
Do Z height alignment
Beam tilt pivot points
Rotation center alignment
Calibrations (if necessary)
Magnification Calibration
Setup Tomography Calibrations
Run Tomography Calibrations
Beam Tilt/Image Rotation
Image Shift
Beam Shift
Induced Beam Shift
Find Suitable Specimen Area
Find feature of interest
Confirm Z Height Alignment
Explore full tilt range
Setup Tomography
Setup Camera
Setup Tomography Parameters
Tilt Angles
Tracking
Memory
Remove backlash in goniometer
Start Data Collection
35
Tomography Setup Checklist
36
Align Microscope
Do Z height alignment
Beam tilt pivot points
Rotation center alignment
Find Suitable Specimen Area
Find feature of interest
Confirm Z Height Alignment
Explore full tilt range
Setup Tomography
Setup Camera
Setup Tomography Parameters
Tilt Angles
Tracking
Memory
Remove backlash in goniometer
Start Data Collection
TEM Tomography Acquisition
Start Tomography
Normally operates automatically
Will request help from user if problem
Several Phases To the Running Tomography Software
Live view
Running
Paused
Manual assist
37
Tomography: Initial Manual Assist
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Tomography: Running
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Tomography: Paused
40
Tomography: Restart
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Tomography: Running
42
Tomography: Manual Assist
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Finishing A Tilt Series
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Tomography Stage Calibration
45
Real Time Measurement Of Stage Drift With
and Without Pre-calibration
46
A Wide Variety Of Electron
Microscopes Are Supported
Need GMS 1.5 or later, current version is GMS 1.7.1
If you have a GIF or a Gatan CCD Camera we support
Most Modern FEI, JEOL and Zeiss TEMs
Many existing microscopes are also supported
Many Phillips CM Series*
Many JEOL FasTEM Series**
Many JEOL Serial Series
Zeiss LEO Series
Need computerized control of stage tilt
Gatan and OEM Holders Supported
Contact Gatan to confirm compatibility
* v12 with low dose is required ** 4 axis motorized stage is required 47
Troubleshooting
48
Contact Information
Where To Get The Latest Version Of DigitalMicrograph?
ftp://ftp.gatan.com/pub/software/PC
Who do I contact?
Field Service
Toll Free +1 888-887-3377
Contact: [email protected]
Western USA Sales
Tel. +1 (925) 463-0200
Contact: [email protected]
Eastern USA Sales
Tel. + 1 (724) 776-5260
Contact: [email protected]
49
Contact Information
Where To Get The Latest Version Of DigitalMicrograph?
ftp://ftp.gatan.com/pub/software/PC
Who do I contact?
Gatan GmbH
Tel. +49 89 358084-0
Contact: mfelsmann @gatan.com
Gatan UK
Tel. +44 1235 540160
Contact: ukinfo @gatan.com
Gatan France
Tel. 33 1 34 94 44 07
Contact: dmonville @gatan.com
Field Service
Toll Free +1 888-887-3377
Contact: [email protected]
50
Working With Tomographic Tilt Series
Import And Export Of Data
Volume Manipulation
Tilt Series Visualization
Integrated 3D Reconstruction
Integration 3D Visualization
51
Import/Export of data
Series can be exported to other data formats through:
File->Save as…
Saves entire 3D cube in another format
Tomography->Export planes…
Saves planes of a series in another format
Series can be imported from other formats through:
File->Open…
Loads a cube saved in another format
Tomography->Import planes…
Loads planes saved in another format
Saving to an external format may lose metadata (tilt angle, scale etc…)
52
VolumeTools
Volume Tools
Provides some capabilities to ‘adjust’ a stack of images, e.g.
Cut off last/first image in a stack
Extract the displayed plane
Accessed on Volume Manipulation sub-menu of Volume
53
Viewing Image Slices
Slice Player
Easy way to ‘play’ a series back and forth
Export playing series to an AVI file
Slice Tool
Control displayed plane
54
Export to AVI
Refine Tilt Series Alignment
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Automatic and
Manual
Alignment
Save offsets
and refine your
alignment later
Available For
Use Offline
3D Reconstruction
WBP and SIRT
Algorithms
Off Center Tilt
Axis Selection
Z Slice
Selection
Integrated
Visualization
Available For
Use Offline
3D Visualization
Volume Visualization
Isosurface
Extraction Tools
3D ROI Tools
Movie Export
Memory Efficient
Available For Use
Offline
Extra Slides After Presentation
58
Troubleshooting: Memory Issues
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Troubleshooting: Memory Issues
60
Digital Micrograph’s Available Memory In 32-bit Windows XP
0 GB 1 GB 2 GB 3GB
Windows XP DLLs
DigitalMicrograph & Plug-ins
Close all other images loaded in DigitalMicrograph
Refer to: http://support.microsoft.com/kb/894472
Gatan can provide you with the Microsoft patch described in this article
Largest Contiguous Block
Image Filtering: Real Space Rectangular
Filter
61
Image Auto-Correlation
Unfiltered
Filtered
Image Filtering: Fourier Space Band Pass
Filter
62
Image Auto-Correlation
Unfiltered
Filtered
Saved Calibrations
63
One Time Microscope Configuration
64
Common PlugIns Installed With
Tomography
65GMS1.6 or earlier Plug-Ins are incompatible with GMS1.7