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 ?