Gatan Webinar Series Automated TEM Tomography...

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

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Principles Of Tomography

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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

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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

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Microscope Preparation

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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

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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

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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

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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

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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

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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

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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

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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

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Camera Setup

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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

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Tomography Setup: Memory

Double check that current tilt series

does not exceed computer memory

Can choose how to save data

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Tomography Setup: General

Save and load previous

tomography parameters

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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

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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

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Tomography: Initial Manual Assist

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Tomography: Running

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Tomography: Paused

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Tomography: Restart

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Tomography: Running

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Tomography: Manual Assist

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Finishing A Tilt Series

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Tomography Stage Calibration

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Real Time Measurement Of Stage Drift With

and Without Pre-calibration

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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

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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: service@gatan.com

Western USA Sales

Tel. +1 (925) 463-0200

Contact: info@gatan.com

Eastern USA Sales

Tel. + 1 (724) 776-5260

Contact: info@gatan.com

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: service@gatan.com

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Working With Tomographic Tilt Series

Import And Export Of Data

Volume Manipulation

Tilt Series Visualization

Integrated 3D Reconstruction

Integration 3D Visualization

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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…)

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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

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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

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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

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Troubleshooting: Memory Issues

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Troubleshooting: Memory Issues

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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

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Image Auto-Correlation

Unfiltered

Filtered

Image Filtering: Fourier Space Band Pass

Filter

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Image Auto-Correlation

Unfiltered

Filtered

Saved Calibrations

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One Time Microscope Configuration

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Common PlugIns Installed With

Tomography

65GMS1.6 or earlier Plug-Ins are incompatible with GMS1.7