10/14/2013
Introduction to
Electron
Microscopy
Alan Nicholls, PhD
Associate Director RRC,
Director Electron Microscopy
Service
Research Resources Center,
UIC
University of Illinois at Chicago
RRC - Electron Microscopy Service
Introduction
Microscopy is one of the few
methodologies applied to nearly
every field of science and
technology in use today.
A microscope can be as simple as a
hand held optical device or as
complex as a multi-million dollar
research multi-million dollar
research tool.
University of Illinois at Chicago
RRC - Electron Microscopy Service
1
10/14/2013
Why do we need microscopy:
• Resolution
of the Human Eye ~ 0.1 mm
• Apparent resolution of the Eye for a Magnified Object is:
R~
0.1 mm
Magnification
Magnification
• 100
• 1,000
• 10,000
• 1, 000,000
Resolvable Distance
1 µm - (1,000 nm)
0.1 µm - (100 nm)
0.01 µm - (10 nm)
0.0001 µm – (0.1 nm)
• What is the difference between Magnification and Resolution?
Magnification: Apparent enlargement of an object
Resolution:
Capability of making distinguishable the
individual parts of an objects
University of Illinois at Chicago
RRC - Electron Microscopy Service
Imaging Resolution
In 1870, Ernst Abbe derived mathematical expression for resolution of
a microscope this was modified by Raleigh.
R = d/2 = kλ
µsinα
k=0.5 Abbe
k=0.61 Raleigh
Center of one Airy disk
is superimposed on the
first minimum of
another (26.5% dip )
Where λ is the wavelength of the
illumination, µ the refractive index of
the lens and α the illumination half
angle. (In light microscopy µsinα is
known as the numerical aperture (NA))
Ernst Abbe
1840 - 1905
University of Illinois at Chicago
RRC - Electron Microscopy Service
2
10/14/2013
Imaging Resolution
In light microscopy the NA of a light optical lens and
therefore resolution can be increased by
a) increasing the half angle (α) of illumination,
b) increasing the refractive index (µ) of the lens by using
Crown glass or oil immersion
or c) decreasing the wavelength (λ) of illumination.
In electron microscopy µ cannot exceed 1.0, α is very
small, and the only thing that can be adjusted is
decreasing the wavelength of illumination, λ, by
increasing the acceleration voltage.
University of Illinois at Chicago
RRC - Electron Microscopy Service
Why Electron Microscopy?
So the main advantage of electron microscopes over light microscopes
lies in the much shorter wavelength of the electron and therefore its much
greater resolution.
Light microscope : UV light, λ is ~400nm >> best resolution (oil immersion) ~ 150nm
Electron microscope: 200kV electrons, λ is 0.0025nm >> resolution ~ 0.015nm
1MV electrons, λ is 0.0009nm >> resolution ~ 0.005nm
Another advantage is that
electromagnetic lenses have
variable focal lengths unlike
lenses used in light
microscopes.
and strength can
be increased by
using a pole
piece to
concentrate the
magnetic field.
University of Illinois at Chicago
RRC - Electron Microscopy Service
3
10/14/2013
Why Electron Microscopy?
BUT
electrons have a short mean free path in air so column has to
be under vacuum this limits the types of specimen we can look
at – nothing WET!
Electromagnetic lenses are not as good as optical lenses and
suffer from aberrations which limits the resolution to ~10x the
Abbe resolution (~0.15nm & 200kV).
University of Illinois at Chicago
RRC - Electron Microscopy Service
Why Electron Microscopy?
Most significant aberration
is spherical aberration, Cs
Light optical lenses Cs
~ µm
Electron optical lenses Cs
~ mm
The shorter the
focal length,
the smaller Cs
and the smaller
the amount of
blur.
Electrons away from the
optical axis are focused to
a different point to those
on axis.
This can now be
corrected, at a price
($1,000,000), in an
aberration corrected
microscope.
University of Illinois at Chicago
RRC - Electron Microscopy Service
4
10/14/2013
Why Electron Microscopy?
BUT
electrons have a short mean free path in air so column has to
be under vacuum this limits the types of specimen we can look
at – nothing WET!
Electromagnetic lenses are not as good as optical lenses and
suffer from aberrations which limits the resolution to ~10x the
Abbe resolution (~0.15nm).
Non-conducting specimens may charge and may need coating
with a conductive film (e.g. C, Au, Cr).
RRC - Electron Microscopy Service
University of Illinois at Chicago
Why Electron Microscopy?
Naked Eye
10-2 m
Natural
World
1 cm
10 mm
Head of a pin
1-2 mm
Ant
~ 5 mm
1,000,000 nanometers =
1 millimeter (mm)
0.1 mm
100 µm
10-5 m
0.01 mm
10 µm
10-6 m
Nanoworld
10-7 m
~10 nm diameter
Pollen grain
Red blood cells
Infrared
10-4 m
10-8 m
Zone plate x-ray “lens”
Outer ring spacing ~35 nm
1,000 nanometers =
1 micrometer (µ
µm)
Visible
Red blood cells
with white cell
~ 2-5 µm
Light optical
microscope
MicroElectroMechanical
(MEMS) devices
10 -100 µm wide
Scanning Electron
Microscope (SEM)
0.1 µm
100 nm
Ultraviolet
Fly ash
~ 10-20 µm
Microworld
Microwave
10-3 m
Man-Made World
Self-assembled,
Nature-inspired structure
Many 10s of nm
0.01 µm
10 nm
Nanotube electrode
ATP synthase
m
Carbon
buckyball
1 nanometer (nm)
Soft x-ray
10-9
Atoms of silicon
spacing ~tenths of nm
10-10 m
0.1 nm
~1 nm
Carbon nanotube diameter
~1.3 nm diameter
Quantum corral of 48 iron atoms on copper surface
positioned one at a time with an STM tip
Corral diameter 14 nm
University of Illinois at Chicago
Transmission Electron
Microscopy (TEM)
RRC - Electron Microscopy Service
5
10/14/2013
Electron Microscopy a brief history
1897 J J Thompson discovers electrons
1924 Louis de Broglie develops theory of wave
nature of electrons
1926 Hans Busch discovers electric fields act as
lenses for electrons
1932 Max Knoll & Ernst Ruska build the first TEM
(x14.4)
1934 TEM with better resolution than LM (x12000)
1938 First Scanning TEM (von Ardenne)
1939 First Commercial TEM, 10nm resolution,
80keV - Siemens, Germany
1940 Commercial TEM with 2.4nm resolution - RCA,
USA
University of Illinois at Chicago
RRC - Electron Microscopy Service
Electron Microscopy a brief history
1942 First Scanning Electron Microscope
1944 First EELS experiments (Hillier)
1954 First lattice resolution image (1.2nm)
~1965 First analytical microscope using XWDS (EMMA)
1966 First Commercial Scanning Electron Microscope (Cambridge S1)
~1970 First commercial energy dispersive X-ray detector (EDAX)
1970 First images of single atoms (STEM, U. Chicago)
1973 First atomic resolution TEM image (0.2nm)
~1977 First commercial EELS spectrometer (Gatan)
1998 First TEM and STEM aberration corrected microscopes
~1999 First commercial Silicon Drift Detector (SDD) for X-rays
2004 First commercial sub-0.1nm resolution (FEI aberration
corrected TEM)
University of Illinois at Chicago
RRC - Electron Microscopy Service
6
10/14/2013
Major Types of Electron Microscope
Scanning Electron Microscope (SEM)
- Beam is scanned across the surface of a bulk specimen and signal
detected from each point using various detectors and displayed on a
monitor.
Transmission Electron Microscope (TEM)
- Beam is transmitted through a sample 3mm in diameter and < 100nm
thick. Image is viewed on a fluorescent screen and collected using a
CCD camera.
Scanning Transmission Electron Microscope (STEM)
- Combination of TEM and SEM using Transmission specimen and
detectors after the specimen. Beam is scanned across specimen.
also known as Analytical Electron Microscope (AEM)
- When combined with detectors for measuring the local composition of
the specimen by X-ray analysis (XEDS) & Electron Energy Loss Analysis
(EELS)
University of Illinois at Chicago
RRC - Electron Microscopy Service
Comparison of types of microscope
University of Illinois at Chicago
RRC - Electron Microscopy Service
7
10/14/2013
Electron Sources
Field Emission Tip
Tungsten Wire
LaB6 Crystal
Thermal Emitters
University of Illinois at Chicago
Thermal
electron
source
RRC - Electron Microscopy Service
What can EM tell us?
Electron Microscopes use a beam of highly energetic
electrons to examine objects at high resolution. They can
yield the following information:
Morphology
The shape and size distribution of the particles making up
the object
Composition
The elements and compounds that the object is
composed of and the relative amounts of them
Crystallographic Information
How the atoms are arranged in the object
Topography
The surface features of an object or "how it looks", its
texture.
University of Illinois at Chicago
RRC - Electron Microscopy Service
8
10/14/2013
Beam Specimen Interaction
The incident electron
beam interacts with the
specimen and produces a
range of different imaging
and analytical signals.
TEM – transmitted and
diffracted electrons.
SEM – secondary,
backscattered and
absorbed electrons
Analytical – X-rays
(XEDS) and inelastically
scattered electrons
(EELS)
University of Illinois at Chicago
RRC - Electron Microscopy Service
Scanning Electron Microscopy
In an SEM:
• The electron beam is focused
to a small spot that is scanned
across the specimen by beam
deflectors
• Electrons that are generated
or back scattered from the
specimen surface, are collected
to form the image
• The specimen can be bulky!
• The image is built up one
pixel at a time, as the beam is
scanned across the specimen
University of Illinois at Chicago
E0 0.1eV-30kV
RRC - Electron Microscopy Service
9
10/14/2013
SEM Imaging Signals
Secondary
Backscattered
Low energy < 50eV
High energy (50% > 0.5Eo)
Atom is ionized by secondary
generation. Relaxation can lead to
generation of a characteristic X-ray
Number backscattered depends on atomic
number of specimen (Z contrast)
University of Illinois at Chicago
RRC - Electron Microscopy Service
SEM Interaction Volume
University of Illinois at Chicago
RRC - Electron Microscopy Service
10
10/14/2013
Scanning Electron Microscopy
Hitachi S-3000N Variable Pressure SEM
0.1-30kV SEM with X-ray. Able to
operate in high vacuum and low
vacuum (1-270Pa), where gas ions
generated by the beam neutralize build up of
negative charge on non-conducting specimens.
Backscattered Electron
Viscoelastic Sponge (20kV, 30Pa, uncoated, backscattered)
University of Illinois at Chicago
RRC - Electron Microscopy Service
Scanning Electron Microscopy
Secondary Electron
Virtually no specimen
prep needed if
specimen is dry and
small enough to fit in
column (6” diameter or
less and less than 1”
high)
Silicon Nanotubes (10kV, uncoated)
Typical XEDS spectrum showing Si, C and O
(10kV) >
University of Illinois at Chicago
RRC - Electron Microscopy Service
11
10/14/2013
Scanning Electron Microscopy
Backscattered Electron
Mouse skin
Split magnification
showing red blood
cells
Wet life science
specimens have to be
fixed (Glutaraldehyde &
and OsO4) and
dehydrated without the
cells collapsing (water
replaced by
hexamethyldisilazane
(HMDS)) and may need
a conductive coating (C
or Au/Pd) to stop
charging.
University of Illinois at Chicago
RRC - Electron Microscopy Service
Transmission Electron Microscopy
The TEM has the same basic
components as a light microscope
• Radiation source (100-300 KeV
electrons)
• Condenser lens
• Specimen
• Objective lens
• Projector lens
• Viewing system
Microscope column is evacuated. Lenses are electromagnetic.
The specimen must be dry or dehydrated and VERY thin
(<100 nm), to permit the electrons travel through the sample.
The entire field of view is illuminated during imaging. The
resulting image is a 2D representation of a 3D object.
University of Illinois at Chicago
RRC - Electron Microscopy Service
12
10/14/2013
Transmission Electron Microscopy
Apertures
University of Illinois at Chicago
RRC - Electron Microscopy Service
TEM Image Contrast
Contrast in the TEM
Nearly all electrons are transmitted through the specimen
in TEM. Contrast arises due to scattering of the beam.
A TEM image is made up of
non-scattered electrons
(which strike the screen) and
scattered electrons which do
not and therefore appear as
a dark area on the screen
Concentrated in smaller angles
Contribute more to analytical signals
Contribute most to image contrast
(dark areas)
Contribute most to image (bright areas)
University of Illinois at Chicago
RRC - Electron Microscopy Service
13
10/14/2013
TEM Image Contrast
Contrast in the TEM
Nearly all electrons are transmitted through the specimen
in TEM. Contrast arises due to scattering of the beam.
Mass-thickness contrast (small objective aperture to select direct
beam)
Dominant contrast mechanism for amorphous specimens.
Diffraction contrast (small objective aperture selecting single spot)
Dominant contrast mechanism in crystalline specimens.
Phase contrast (large objective aperture to select multiple spots)
Contrast is due to the phase of the electron wave at the exit
plane on the specimen.
University of Illinois at Chicago
Mass-thickness contrast
Atrial Muscle (80kV)
Contrast is enhanced by
staining with heavy metals
(U, Pb) which are taken up
non-uniformly by the
specimen. Dominant in
amorphous specimens.
University of Illinois at Chicago
RRC - Electron Microscopy Service
Diffraction contrast
Stacking faults in Al
Bragg scattering from crystalline
planes in specimen. Dominant in
crystalline specimens
RRC - Electron Microscopy Service
14
10/14/2013
Phase contrast
Dislocation in Silicon
“Atomic” resolution images can be
obtained, close to focus, on oriented
crystalline specimens due to elastic
scattering of the beam by the atomic
nuclei. Contrast due to phase of
electron wave at exit plane of specimen.
Diffraction Pattern
Tin oxide nano-crystals
Changing projector lens strength a
diffraction pattern can be displayed
instead of an image
University of Illinois at Chicago
RRC - Electron Microscopy Service
Transmission Electron Microscopy
JEOL JEM-3010
300kV Materials Science TEM with
Gatan CCD camera for imaging and
Noran X-ray detector for
microanalysis.
Atomic resolution
image of
polycrystalline
Barium Titanate
(300KeV)
University of Illinois at Chicago
RRC - Electron Microscopy Service
15
10/14/2013
TEM Specimen Preparation
Preparation can
take from minutes
(particles) to days
(cross sectional)
University of Illinois at Chicago
RRC - Electron Microscopy Service
TEM Specimen Preparation
Specimens are nearly always circular and 3mm in diameter.
Perpendicular to this disk, the specimen must be thin enough for
electrons to pass through (t<100nm).
Self supporting, electrochemically
thinned to perforation, Al-4% Zn
University of Illinois at Chicago
Spore supported on a carbon film
on a locator grid
RRC - Electron Microscopy Service
16
10/14/2013
TEM Specimen Holder
Single tilt
specimen
holder
Tip is removable and is
supported while the
specimen is loaded
University of Illinois at Chicago
RRC - Electron Microscopy Service
Transmission Electron Microscopy
Er coated Si nanowire showing faults
in the Si nanowire and crystallinity in
the Er coating.
dispersed onto a carbon film (300KeV)
Dark Field 1
Dark Field 2
Bright Field
DF1
DF2
University of Illinois at Chicago
RRC - Electron Microscopy Service
17
10/14/2013
Transmission Electron Microscopy
SiGe quantum well
srtructure at atomic
resolution - self
supporting specimen
110
diffraction
pattern
from area
DF1
DF2
University of Illinois at Chicago
Low mag
of similar
area
RRC - Electron Microscopy Service
Analytical Electron Microscopy
JEOL JEM-ARM200CF
200kV Cold Field Emission Probe
Aberration Corrected Materials
Science TEM/STEM with Gatan Enfina
Electron Energy Loss Spectrometer
and Oxford SDD X-ray detector for
microanalysis.
University of Illinois at Chicago
RRC - Electron Microscopy Service
18
10/14/2013
Analytical Electron Microscopy
A TEM becomes an Analytical Electron Microscope (AEM) when
spectroscopic information can be collected as well as images.
Typically X-Ray (XEDS) and Energy Loss (EELS). AEMs will
often be capable of operating in Scanning Transmission (STEM)
mode.
In a field emission STEM the probe size can be smaller than
interplanar spacing. Some electrons suffer a near nuclear
collision and are elastically scattered through large angles.
When collected by a high angle annular detector, the image not
only contains atomic resolution, but the intensity of the atom
columns is proportional to Z.
Using the annular dark field detector allows simultaneous
acquisition of the EELS spectrum.
University of Illinois at Chicago
RRC - Electron Microscopy Service
Analytical Electron Microscopy
Incident Probe
Annular
Detector
Z-contrast
Image
BF-Image
Spectrometer
CCD-EELS
Detector
University of Illinois at Chicago
RRC - Electron Microscopy Service
19
10/14/2013
Analytical Electron Microscopy
Inelastic Scattering
(fast electron – electron)
Elastic Scattering
(fast electron – nucleus)
XEDS
HAADF
STEM
Mass/ Thickness
EELS
Diffraction
University of Illinois at Chicago
Spectroscopy
RRC - Electron Microscopy Service
Analytical Electron Microscopy
Atomic resolution HADF
STEM image of a bismuth,
strontium, copper, calcium
oxide specimen containing a
fault. The heaviest atomic
number element (Bi) is the
brightest with darker Sr atoms
on each side of the Bi. In
between there are three Cu/Ca
columns.
In the faulted region there are
two more Cu/Ca columns but
the structure recovers within
two unit cells by loosing a
Cu/Ca column.
University of Illinois at Chicago
RRC - Electron Microscopy Service
20
10/14/2013
AEM – X-ray analysis
Sum
Sumspectra
spectrafrom
fromrectangular
circular area
area
X-Ray
Spectrum
Imaging
Collecting full
spectrum from
every point in
scanned image
Data can be
interrogated
after collection
University of Illinois at Chicago
RRC - Electron Microscopy Service
AEM - EELS
Z contrast image showing
atomic core structure of
mixed dislocation. EELS
from core and bulk showing
increase in first peak
Z contrast image of partial dislocation. EELS
spectra from various positions on partial
dislocation compared to bulk showing differences
in electronic state
University of Illinois at Chicago
RRC - Electron Microscopy Service
21
10/14/2013
Aberration Corrected STEM
Uncorrected,
Cs=0.5mm
JEM-2010F 0.13nm probe size,
Si <110>, Dec 1998
Corrected, Cs=-0.5µm
JEM-ARM200CF 0.08nm probe
size, Si <110>, Oct 2011
There are similar analytical improvements
University of Illinois at Chicago
RRC - Electron Microscopy Service
Aberration Corrected STEM
Pt on Alumina catalyst
Catalytic performance
dependent on Pt particle size.
Now able to image individual
platinum atoms
What effect do these have on
catalytic properties?
Visible also on carbon film, not
as attracted to substrate as
clusters? Are they present on an
active catalyst after use?
Higher resolution answers some
questions but results in many
more!
University of Illinois at Chicago
RRC - Electron Microscopy Service
22
10/14/2013
Aberration Corrected STEM
Atomic-resolution Z-contrast
image of epitaxial LaCoO3 films
grown in LaAlO3 showing a
superstructure.
Z-contrast images of SrTiO3 film on GaAs for
two different growth conditions. The SrTiO3 is
shown in the [001] orientation while the GaAs
is seen along the [110] in both samples. a):
SrTiO3 film deposited on GaAs after depositing
a Ti pre-layer; b): same growth conditions
forSrTiO3 except that no Ti pre-layer was
deposited.
University of Illinois at Chicago
RRC - Electron Microscopy Service
Aberration Corrected STEM
Z-contrast image of Ca3Co4O9 [110]
Simulated HADF
ABF of Ca3Co4O9 [110]
Simulated ABF
Atomic Resolution EELS
Imaging
Ca L (green), Co L (blue), O K (red)
R.F. Klie, Q. Qiao, T. Paulauskas, Q. Ramasse, M.P. Oxley, J.C. Idrobo, Phys. Rev. B, 85(5), 054106 (2012)
University of Illinois at Chicago
RRC - Electron Microscopy Service
23
10/14/2013
Aberration Corrected STEM
Atomic
Resolution
XEDS Imaging
University of Illinois at Chicago
RRC - Electron Microscopy Service
The Need for 3-D Analysis
A two-headed
rhino – a
projection artifact
A TEM produces a
2-D image of a 3-D
object
Hayes (1980) – “When we see this image we laugh, but when we see
equivalent (but more misleading) images in the TEM, we publish!”
University of Illinois at Chicago
RRC - Electron Microscopy Service
24
10/14/2013
The Need for 3-D Analysis
By looking only in projection we can be fooled!
>> Electron Tomography
RRC - Electron Microscopy Service
University of Illinois at Chicago
Tomographic Reconstruction
Representation of sampling in
Fourier space
Data
points
(b) Weighted backprojection
– to account for under
sampling of high frequencies
ORIG
High frequencies
undersampled
WBPJ
BPJ
(c) Iterative backprojection (SIRT) – iteratively
constrain the reconstruction to be give re-projected
images that are identical to the original projections.
The Principle of
(a) Backprojection
0
2
4
6
8
10
P. A. Midgley and M. Weyland, Ultramicroscopy 96, 413-431 (2003)
University of Illinois at Chicago
RRC - Electron Microscopy Service
25
10/14/2013
Tomographic Reconstruction
Catalyst tilt series
Pt on SiO2
One axis tilt
through ±60o
Images acquired
every 1.5o
University of Illinois at Chicago
RRC - Electron Microscopy Service
In Situ – Heating/ Cooling Stages
Gatan 652
double tilt
heating stage
Gatan 636
double tilt
LN2 cooling
stage
University of Illinois at Chicago
RRC - Electron Microscopy Service
26
10/14/2013
In Situ – SPM inside the TEM
SPM Probe
Specimen
SPM Probe
Nanofactory STM-TEM stage
Specimen
SPM probe being bought into contact
with nanowire, eventually voltage
applied which melts nanowire
University of Illinois at Chicago
RRC - Electron Microscopy Service
Conclusions – Electron Microscopy
• Electron microscopy is a powerful tool for the study of materials.
• Transmission EM is a full field viewing technique, like conventional light
microscopy
• Scanning EM and STEM are point scanning techniques
• Specimen preparation techniques are often complex and timeconsuming especially for cross sectional materials microscopy.
• The higher the resolution, the poorer the sampling. A 1970 estimate was that
all TEMs in the world from 1940 to 1970 had only looked at 0.3mm3 of material.
• SEM images are of the surface of the specimen.
• TEM & STEM images are 2D images of 3D objects – generally all TEM
information is averaged through the thickness of the specimen.
• Ionizing radiation can damage the specimen. Contamination from
hydrocarbon build up can occur – specimens should be as clean as
possible
• Is what you see representative of the whole specimen?
University of Illinois at Chicago
RRC - Electron Microscopy Service
27
10/14/2013
Electron Microscopy Service @ UIC
JEM-1220
AXIS-165 XPS
JEM-3010
JEM-ARM200CF
VT-SPM
University of Illinois at Chicago
HB601UX
Ramascope 2000
JSM-6320F
S-3000N
RRC - Electron Microscopy Service
28