Download Introduction to electron microscopy Transmission electron

Introduction to electron
microscopy
py
NANOTEM Lecture Series
Characterization of materials
Arto Koistinen, M.Sc.
BioMater Centre
23.11.2009
Transmission electron
microscope (TEM)
1
"Short history"
Louis de Broglie in the early 1920's: a theory of particles
having wave-like properties
In the 1920's: Schrödinger ja Heisenberg developed a
theory of quantum mechanics
mechanics, which "enabled" electron
microscopy
In 1926 H. Busch proved mathematically that electrons can
be focused by a magnetic field with the similar way as light
is focused in an optical lens
Ernst Ruska developed a lens system able to magnify
specimen by 16x! (Published in 1931; they used a term
'electron microscope')
p )
R. Ruedenberg (working for Siemens) applied a patent and in some
references he has been mentioned as the inventor of EM.
In1939 the first TEM was manufactured (by Siemens)
In1986 Ruska was awarded with the Nobel Price
Transmission
electron
microscope,TEM
Ultra thin slices of specimens or very small particles
are investigated.
The principle of operation:
2
Structure of TEM;
TEM vs. LM (Light microscope)
In fact, the
microscopes are
pretty similar!
Sample preparation for TEM
Sample preparation is the most critical part in EM
studies!!!
Special equipment and skillful technicial are needed
Biological samples for TEM need…
fixation
dehydration
embedding
cutting
staining
Notes:
sample size at final state < 1 mm
typical slice thickness about 50 nm
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Example: TEM sample preparation
UNIVERSITY OF KUOPIO
BioMater Centre
BASIC METHOD FOR ANIMAL TISSUES, phosphate buffer
Pre fixation:
Pre-fixation:
- perfusion fixation and/or immersion fixation
- 2 % glutaraldehyde in 0,1 M phosfate butter, pH 7,4
Rinsing:
- 0,1 M phosfate buffer, pH 7,4
Post fixation:
- 1 % osmiumtetraoxide (OsO4) in 0,1 M phosfate buffer, pH 7,4
Rinsing:
- 0,1 M phosfate buffer, pH 7,4
Dehydration:
- 70 % ethanol
- 90 % ethanol
- 94 % ethanol
- abs. ethanol
- propyleneoxide
- propyleneoxide
Infiltration:
- Mix of propyleneoxide and LX-112 1:1
- LX-112
Embedding:
- fresh LX-112, embedding in appropriate molds
Polymerization:
- 37°C
(in heat oven)
- 60°C
110
2-4 h
15 min
2h
15 min
10 min
10 min
10 min
3 x 10 min
15 min
10 min
2h
overnight
24 h
48 h
Note: This takes 4-5 days!
Still cutting (with diamond blade) and staining with heavy element salts are needed.
Some examples
LM images
TEM images
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Sample preparation for TEM:
"Hard samples"
Ion beam milling is used
Operation of TEM;
Basics of image formation
Part of the beam electrons hit the nuclei or electrons of
the atoms in specimen,
p
, i.e. theyy are scattered
Scattered electrons are cropped by using apertures
Dense sections in the specimen (i.e. stained parts)
cause more scattering and are dark in the image plane
The most important factor in image formation in TEM
is scattering
g
(NOTE! In light microscopy; absorbtion)
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Structure of TEM 1;
Cross-section of the equipment
Structure of TEM 2;
"Electron gun"
Electron source ("gun")
Electrons are emitted from a tungsten filament (thin wire)
Also modern types of guns are developed with higher stability,
longevity and brightness; LaB6 and field emission
Electrons are accelerated with an electric field (80 kV or 200
kV, for example) towards the specimen
"Electron gun"
"Properties of guns"
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Structure of TEM 3;
Lens system
Lens system
All lenses are electromagnetic lenses
Electrons can be controlled by the
magnetic field
Firstly, electron beam is focused to
the sample by condensor lenses
Objective lens (after the sample)
forms an image of the specimen
Intermediate lenses and projector lens
magnify
g y the image
g
Image recording system
Nowadays, the image is recorded by a
CCD camera
(or still by using plate films)
Basics of microscopy
Resolution (r, "resolving power")
r
Resolving power is the minimum distance between two spots
that can be seen as individual spots
Human eye: 0.1 mm = 100 μm = 100000 nm
Light microscopy: 0.0002 mm = 0.2 μm = 200 nm
Electron microscopy: 0.0000001 mm = 0.0001 μm = 0.1 nm
Human eye
silmä
Light
microscopy
valomikroskooppi
l ik
k
i
Transmission
electron microscopy
läpäisyelektronimikroskooppi
0
1
10
100
0,01
0,1
1000
1
10000
100000
10
100
0,01
0,1
1000000 nm
1000
1
um
mm
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Basics of microscopy;
Resolving power
Resolving power…
depends on the wavelength of the light
is roughly half of the wavelength
For example; Using visible light (n. 400 – 700 nm) the resolution is
about 200 nm at maximum
"Behind the scenes":
r=
Formed
image
Point
source
0.612 ⋅ λ 0.612 ⋅ λ
=
n ⋅ sin
i α
N . A.
where, λ
= wavelength,
= refractive index,
α = angle in the lens system,
N.A. = numerical aperture
n
Diffraction
in the slit
or aperture
Basics of microscopy;
Resolving power (TEM)
Also motion of the electrons
include wave-like behaviour
(theory by de Broglie), and
the wavelength depends on
the acceleration voltage:
„
Acceleration
voltage (kV)
Wavelength (nm)
10
0.0122
50
0.0054
100
0.0037
1000
0.0009
"Behind the scenes":
Energy of particle = Energy of quantum:
… de Broglie wavelength can be calculated:
…
…
Speed of electrons can also be calculated
E = mc 2 = hc / λ
h
h = Planck's constant
λ=
m = electron mass
mc
c = speed of light
(assuming energy from acceleration = kinetic energy of the particle):
eV =
…
1 2
mv
2
⇒
v=
2eV
m
e = electron charge
V = acceleration voltage
v = speed of electron
NOTE! With acceleration voltage 50 kV the speed of the electrons is
about 15 % of the light speed --> theory of the relativity has to be
considered
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Some examples 2
Capillary
(scale bar 2 μm)
Bacteria
(scale bar 0.2 μm)
"Dust particles"
(scale bar 50 nm)
Modern techniques:
Tomography with TEM
3D--object => set of 2D
3D
2D--projections
2D--projections => 3D
2D
3D--reconstruction
S. Nickell, C. Kofler, A. Leis, W. Baumeister: Nature Reviews Molecular Cell Biology
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Example of tomography:
3D organization of organelles in cells
Murk et al. Traffic 2004; 5: 936-945
Different types of MLLs. A)
Tomographic slice (resolution
of 4nm) of 250nm section
showing the concentric
o g ni tion of inte
organization
internal
n l
membranes in a high-pressure
frozen hDC. B) MLL in high
pressure frozen B -lymphocyte
containing membrane sheets
and small vesicles. C, D) 3-D
model of internal membranes
with an onion-like organization
of vacuoles present in MLL
shown in A.
Scanning electron
microscope, SEM
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"Short history"
Developed by M. Knoll in 1935;
Patented by M. von Ardenne in 1937
The first commercial SEM in 1965
Cambridge Scientific Instruments: Mark I
This was a breakthrough of electron microscopy, because SEM was
found to besuitable in various applications
Note! TEM was developed earlier in the 1930's
In the end of 1960's, elemental analysis attached (WDS)
Thereafter, methodological and technological development
have improved the performance
For example;
electron source stability --> better resolution,
vacuum systems --> different imaging modes,
information technology --> data storage and manipulation
Nowadays, SEM if by far the most common type of
electron microscopes
Basics
Surfaces and surface related structures,
topography and morphlogy of the specimens
are investigated with SEM
Basic components in the equipment:
Electron source, vacuum system, magnetic lenses
and signal detection unit
Note! Can you define SEM as a microscope?!?!
SEM, Philips XL30
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Operation of SEM;
SEM vs. TV
Electron gun
Lenses
All are
condensing
Deflector
Scanning
Detector
"P l meter"
"Pulse
t "
Visualization
Sample preparation for SEM;
Basic requirements
Samples must fulfil the basic requirements:
1 - Must fit in the specimen chamber and the holders
2 - Stability;
- no evaporation of liquids is allowed
- sample must remain unchainged in electron bombing
--> Risk of contamination and structural changes
3 - Conductivity; charging of the sample creates gives
poor results
- Coatings,
Coatings low acceleration voltage or special euipment
prevent the problem
4 - Cleanliness;
- dirt on the sample may interfere the investigation
--> Note: sometimes the "dirt" is being investigated
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Charging / stability
Charging of the sample
Damage due to electrons
Examples
Metallic screw
(untreated, SEM mode)
„
Polymeric implant
(untreated, low vacuum mode)
„
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Sample preparation for SEM
Again, sample preparation is critical in SEM studies
Special equipment and reagents are typically used
Biological samples for SEM need…
fixation
dehydration
coating
(e.g. critical point drying)
(sputter coating with Au or Pt)
Sample preparation for SEM;
effect of fixation method
Physical fixation
(fro en and fractured)
(frozen
fract red)
Chemical fixation
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Sample preparation for SEM;
effect of drying method
Sample preparation for SEM:
"Hard samples"
Ion beam cutter
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Operation of SEM;
Image formation
High-energy beam electrons hit the atoms in specimen and
thus, secondary electrons are scattered from the
specimen and detected.
detected
Note! Beam electrons have energy 2- 30 kV, whereas the detectable
electrons (secondary electrons) have energy only about 10-20 eV
Examples: Biological samples
Pollen:
Cultured cells:
Bacteria:
Red cells:
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Operation of SEM;
Beam/specimen interaction
Due to electron bombing
different types of particles
or radiation is emitted from
the sample
These signals can be
detected and used for
characterization
Resolution of the signals are
proportional to the interaction
volume
Note: For imaging, the
resolution can be < 1 nm!
Imaging with SEM:
Effect of acceleration voltage
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Other imaging modes with SEM: BSE
Backscattered electrons (BSE):
BSEs are beam electrons which escape
from the specimen --> BSEs have higher energy than SEs
Information acquired with BSEs:
Depth-related structural information
Info of chemical composition
Backscattered Electron Image
BSE, 10 kV
BSE, 3.5 kV
Other modes of SEM:
Low vacuum -mode
Used for imaging of non-conductive samples
polymers,
p
y
, biological
g
samples...
p
Relative humidity in the chamber is raised, and ionized gas
molecules transfer excessive electrons to prevent charging
Additional GSE-detector is required
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Other modes of SEM:
Environmental SEM (ESEM)
Relative humidity and temperature can be
controlled
--> solid/liquid phases
--> swelling, etc.
An example: salt crystals
Modern techniques:
Tomography with SEM
Principle:
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Example of tomography with SEM
A ceramic sphere containg bubbles.
Sphere diameter 90 microns.
Data courtesy of Dr Sherry Mayo
THANK YOU!
For more information, please visit
http://www uku fi/biomater
http://www.uku.fi/biomater
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