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Gatan's expert Neil Wilkinson presented at ETC Environmental and Bio-Sciences TEM Open Day
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Brunel TEM Presentation
Some applications of TEM for Biological Applications
Neil Wilkinson Gatan UK
June 2013
Introduction Basic TEM Imaging Specimen Prep. What is EELS, GIF, EFTEM, spectroscopy etc Electron Specimen Interaction GIF – The way it works EFTEM & Spectroscopy Infected Blood Cell data
Specimen Prep Sectioning Particulates Dried samples Cryo Sectioning
Image examples
ORIUS 1000 CCD camera
ES1000 Skeletal Muscle
ORIUS SC1000B: HREM image of GaSb/InAs superlattice
Image courtesy of C.H. Lei and J.G. Wen at the Center for Microanalysis of
Materials (CMM) at the University of Illinois, Urbana-Champaign, USA
Electron Diffraction
Low Dose Cryo – special cameras
Low Dose cryo image (part of 4k image) GRO EL protein.
Special Low Dose Scintillator – High “conversion factor”
Low Dose software on microscope essential.
Typically 10 – 20 e/Å dose to avoid damage.
Ideal camera – high resolution & VERY large area.
1000’s of “particles” per image.
10k x 10k best available.
Price !!!!!!!!!!!!!!!!!
Filtered Image for best resolution and contrast – GIF Zero Loss Imaging.
First CCD only reconstruction 14Å Subramanian et al Journ Struct Biol 143 (2003)135-144
CCD reconstruction now better than 5 Å
Current best
resolution
Reconstruction
3.3Å
Special New
Camera
Cryo Prep
Exceeds X-Ray
Resolution
“Direct Detection”
TEM AUTOMATION – using CCD.
• Focus
• Astigmatism correction
• Alignment
• Digital Montage
• Unattended Image capture – Automated - 10’s of thousands of images from Multiple samples.
• Tomography
• EELS, SFTEM, etc.
TEM Tune - Example
Focus Stigmation Alignment
*
*
TEM Automation - DigitalMontage
Unstained
liver sample
3 x 3 automated
MONTAGE
TEM Automation - DigitalMontage
Montage of 9
Images
EELS/EFTEM/STEM/STEM EELS SI
Electron Energy Loss Spectrometry – EELS
Gatan Image Filter – GIF
Energy Filtered TEM – EFTEM
Scanning Transmission Electron Microscopy – STEM
STEM EELS Spectrum Imaging – STEM EELS SI
beam-specimen interactions and signals
Elastic scattering (Diffraction)
X rays (EDS)
Auger electrons
Backscattered electrons
Elastic & Inelastic scattering (EELS)
Secondary electrons
Thin TEM Specimen
+
e-
e-
e-
e-
e-
e-
e- e- e-
elastically scattered e-
in-elastically scattered e-
un-scattered
19
The EELS Spectrum – Main feature – Zero Loss
20
The EELS Spectrum – Main features – Zero Loss & Core Loss
Elastic Inelastic
21
The EELS Spectrum – Main features and uses
In-Column & Post Column Filters
GIF – Imaging Mode
23
Vertically dispersed spectrum formed near back focal plane of prism
GIF – Imaging Mode
24
Unfiltered TEM
Image mode
Unfiltered image projected onto CCD detector
GIF – Imaging Mode
25
Core-loss EFTEM
Image mode
Energy-selecting slit inserted
Core-loss image projected onto CCD detector
GIF – Spectroscopy Mode
26
Low-loss EELS
Spectrum mode EELS spectrum projected onto CCD
Final EELS readout
GIF – Spectroscopy Mode
27
Core-loss EELS
Spectrum mode
Spectrum offset via prism current or high tension
EELS spectrum projected onto CCD
Final EELS readout
O K edge
Mn L edge
28
• EELS provides another dimension for generating image contrast
• EELS isolates elastic scattering for enhanced imaging
170 eV, contrast inverted Unfiltered bright-field TEM Loss image at 170 ± 15 eV
Contrast enhancement - inelastic only
Most Probably Loss Imaging
• Minimum of two background windows are necessary
• Map intensity is directly proportional to projected concentration
• Map-intensity can be related to
absolute concentration if thickness and elemental cross-sections are known
Elemental mapping – 3 Window or Jump Ratio
Determine and subtract edge background using two pre-edge images to obtain the
true edge signal
EFTEM: 3-window mapping
3
2 32
Pre edge 1 (485eV) Pre edge 2 (515eV) Post edge (542eV)
Elemental map Jump ratio image
Semiconductor
O-N-O layers
Oxygen Maps
EFTEM Elemental mapping vs. jump ratio imaging
Benefits of resolving energy loss in ETEM - Imaging
• EELS can be used to identify and map elements and phases
3
3
Each 3 window EFTEM
map takes approx 20 – 30
seconds to collect.
STEM spectrum image acquisition
3
4
34
Acquired by stepping a focused electron probe from one pixel to the next
EDX
STEM
EELS DF
Specimen
Spectrum image data cube is filled one spectrum column at a time
STEM allows collecting any combination of EELS, x-ray, and CL spectra simultaneously
DF or SE signal permits spatial drift correction during acquisition
x
y
E
What is Spectrum Imaging?
• Collects detailed spatial and spectroscopy information • Allows processing decisions after acquisition
• Spectrum imaging can create quantitative images / profiles
• Can confidently locate artifacts & understand image contrast
x, y spatial dimensions
E energy-loss dimension
y
x
E
image at E1
image at E2
image at Ei
.
.
.
.
.
.
.
.
.
spectrum at xi , yi
3
6
STEM/EELS Spectrum Image – “Data Cube”
STEM/EELS Spectrum Imaging – extraction of maps
3
7
37
Elemental maps extracted from STEM/EELS “Data Cube”
Si Al
Ti N
W SiO2
Summary
• Very Well equipped TEM/STEM with many high end accessories
• Conventional TEM Imaging
• TEM Tomography
• STEM HAADF Imaging
• EFTEM – Energy Filtered Imaging
• EELS – Electron Energy Loss Spectroscopy
• STEM/ EELS – EELS Mapping
Any Questions ?