1. No category

Sample Preparation
for
Transmission Electron Microscopy
Holm Kirmse
Humboldt University of Berlin
Institute of Physics
TEM Group
Outline
• Motivation
• Criteria for appropriate preparation
• Preparation techniques and applications
• Layered structures
• Bulk material
• Particles
• Summary
Why sample preparation for
Transmission Electron Microscopy
• Electrons with properties of particles and waves
• Strong interaction between electrons of the beam and
atoms of the samples  scattering
• Sufficient intensity/number of transmitted electrons
only for small thickness (about 100 nm)
• Essential thickness depends on, e.g.,
materials properties, acceleration voltage, and
requirements of individual investigation method
Scattering of electrons
Bulk material
TEM specimen
50 nm
1 nm
50 µm
200 nm
Monte-Carlo Simulation of the paths
of electrons (acceleration voltage: 100 kV)
impinging Si for different thicknesses
Full width at
half maximum
12 nm
Demands on sample preparation
• No change of materials properties including:
– Structure (amorphous, polycrystalline, crystalline)
– Chemistry (composition of the bulk material,
of surfaces, and of interfaces)
• But:
Artifacts inherent to any preparation method!
• Criterion for appropriate preparation technique:
Influence on structural and chemical properties
as little as possible!
Type of sample
• Layered structures
• Bulk material
• Particles
not transparent for electrons
transparent for electrons
size < 100 nm
Shape of the sample
• Defined by TEM sample
holders
• Limits of sample size:
– Diameter:  3 mm due
to the furnace of the
TEM sample holder
– Maximum thickness of
sample edge:
ca. 100 µm
Aim of investigation
• Structural properties
– size distribution of
entities
– area density
– structural defects
• Chemical properties
– composition
– modification
– interface sharpness
• Electronic properties
• Magnetic properties
Type of sample
• Bulk material
• Layered structures
• Particles
Materials properties
• Hardness
• Sensitivity for chemical
solutions
Preparation strategy
Strategies of TEM preparation
DEPOSIT
evaporate
sputter
BULK
>3 mm
SURFACE/
INTERFACE
>3 mm
SMALL PIECES
>3 mm
PLAN
protect on
one side,
shield
lacquer
CROSS-SECTION
build up to
1.5 mm depth,
plate deposit embed,
glue face to face
FILM/
GRID
INITIAL
PREP.
cut,
lap,
grind
D
I
M
P
L
E
JET
electropol.
chem-pol.
ion mill
DISK
SHEET
cut
HAND
electropol.
chem-pol.
FOIL/
GRID
BLOCK
cut
ULTRA
MICROT.
SECTION/
GRID
ETCH
acid
REPLICATE
+ release etch?
EXTRACTION
REPLICA/
GRID
DISK
cut, punch, drill
M
I
R
O
S
C
O
P
CRUSH
pestle,
mortar
CLEAVE
razor,
blade
C
E
FRAGMENTS
SUPPORT FILM/
GRID
Layered Structures
Strategies of TEM preparation
DEPOSIT
evaporate
sputter
BULK
>3 mm
SURFACE/
INTERFACE
>3 mm
SMALL PIECES
>3 mm
PLAN
protect on
one side,
shield
lacquer
CROSS-SECTION
build up to
1.5 mm depth,
plate deposit embed,
glue face to face
FILM/
GRID
INITIAL
PREP.
cut,
lap,
grind
D
I
M
P
L
E
JET
electropol.
chem-pol.
ion mill
DISK
SHEET
cut
HAND
electropol.
chem-pol.
FOIL/
GRID
BLOCK
cut
ULTRA
MICROT.
SECTION/
GRID
ETCH
acid
REPLICATE
+ release etch?
EXTRACTION
REPLICA/
GRID
DISK
cut, punch, drill
M
I
R
O
S
C
O
P
CRUSH
pestle,
mortar
CLEAVE
razor,
blade
C
E
FRAGMENTS
SUPPORT FILM/
GRID
Example of scientific problem
GaSb / GaAs Islands (Quantum Dots)
GaAs
3.6 ML
4.0 ML
GaSb
GaAs
4.5 ML
5.0 ML
5.4 ML
5.8 ML
GaAs
Photoluminescence
3 mm
Layered structures
Plan view
Cross section
3 mm
Layered structures
Plan view
•
•
•
•
Area density
Lateral size
Lateral correlation
Morphology
Cross section
• Height
• Width
• Be aware of
projection artifacts!!!
Plan-view and cross-sectional
TEM preparation
Plan view (PVTEM)
Cross section (XTEM)
Initial sample
Formatting
Thinning of substrate
face-to-face gluing
Gluing in a cylinder
and sawing
Mechanical thinning
Cutting of a disc
Gluing of dummies
Dimpling
Dimpling
Ultrasonic disc cutting
Ion-beam milling
Ion-beam milling
Formatting
Face to face gluing
sample
Wire saw with wire of steel
covered by diamond particles
Gluing into a cylinder
and cutting into discs
Ultrasonic
cutter
Mechanical thinning
Grinding and polishing tool:
1 - silicon nitride pads,
2 - body part, 3 - screw
Cross sectional specimen after
mechanical polishing
In general: first remove the damaged layer of the previous step
Mechanical thinning  damage
Region 1
Beilby layer: change of
chemical composition,
strong deformation,
amorphization
Region 2
macro-deformed layer:
tilt of grains,
increased dislocation density
Region 3
micro-deformed layer:
weak tilt of grains,
dislocation density as grown
~ 1000 nm
Situation after sawing
Next preparation step has to remove the damage!
Dimple grinding
Detail of a dimple grinder
sample
Dimpler grinder of Gatan
Principles of dimpling technique
Ion-beam milling
sample
Ion gun arrangement for milling of
both sides of the sample;
possible ions: Ar+, Xe+, I+, ...
acceleration voltage: 1...5 kV
usual angle g: < 10°
Layout of a vacuum chamber
with two ion guns
Ion-beam milling machines
a)
b)
c)
a) Precision Ion Polishing System PIPS (Gatan);
b) and c) two versions of the Rapid Etching System RES (Leica)
Cross-sectional TEM imaging
Strain-sensitive image contrast
Chemical sensitive image contrast
Plan-view TEM imaging
5.5 ML
5.0 ML
defect
4.0 ML
3.6 ML
41010 cm-2
31010 cm-2
11010 cm-2
GaSb/GaAs
Growth interruption time: 5 s
Size distribution of GaSb/GaAs QDs (a)
and corresponding PL spectra (b)
a)
GaSb layer thickness
b)
4.0 ML
5.0 ML
10
3.6 ML
5.5 ML
4.0 ML
number ∙ 109 / cm²
8
6
4.5 ML
4
5.0 ML
5.4 ML
5.8 ML
2
0
0
10
20
30
d / nm
40
50
Growth interruption time: 5 s
Focused-ion beam thinning
FEI FIB Strata 201
Ion getter
pump
Ion
source
Camera
Aperture
SE
detector
Stage
Focussed-ion beam preparation
of a device structure
Pt deposition
Terrace cut
Cleaning I
Cleaning II
U cut
Transfer to TEM grid
Comparison of XTEM preparation
Conventional procedure
Wire-shadow
technique
Focussed ion-beam
method
Comparison of conventional XTEM
and FIB preparation technique
FIB
+ high position accuracy
+ time consume about 2.5 h
– radiation damage
– high costs
Conventional preparation
+ low radiation damage
+ low costs
– time consume about 30 h
– low position accuracy
Bulk Material
Strategies of TEM preparation
DEPOSIT
evaporate
sputter
BULK
>3 mm
SURFACE/
INTERFACE
>3 mm
SMALL PIECES
>3 mm
PLAN
protect on
one side,
shield
lacquer
CROSS-SECTION
build up to
1.5 mm depth,
plate deposit embed,
glue face to face
FILM/
GRID
INITIAL
PREP.
cut,
lap,
grind
D
I
M
P
L
E
JET
electropol.
chem-pol.
ion mill
DISK
SHEET
cut
HAND
electropol.
chem-pol.
FOIL/
GRID
BLOCK
cut
ULTRA
MICROT.
SECTION/
GRID
ETCH
acid
REPLICATE
+ release etch?
EXTRACTION
REPLICA/
GRID
DISK
cut, punch, drill
M
I
R
O
S
C
O
P
CRUSH
pestle,
mortar
CLEAVE
razor,
blade
C
E
FRAGMENTS
SUPPORT FILM/
GRID
TEM preparation of bulk materials
analogous to
plan-view preparation
of epitaxial structures
Plan-view and cross-sectional
TEM preparation
Plan view (PVTEM)
Cross section (XTEM)
Initial sample
Formatting
Thinning of substrate
face-to-face gluing
Gluing in a cylinder
and sawing
Mechanical thinning
Cutting of a disc
Gluing of dummies
Dimpling
Dimpling
Ultrasonic disc cutting
Ion-beam milling
Ion-beam milling
Cleaving
Requirement:
Proper cleavability
Comparison of samples prepared by
cleaving or ion milling
ZnSe
GaAs
Cleaving:
 few artifacts
- large angle
at the edges
Ion milling:
- artifacts in ductile material
 low angle  large area for
investigation
Ultramicrotome cutting
Slicing of the specimen
embedded in epoxy or other
medium
Floating of the slices onto water
or an appropriate inert medium
and collection on TEM grids
Ultramicrotomy vs. ion milling
Ultramicrotomy, cut along the HIOS interface
HRTEM, slit width 10 eV, df = -1000 nm
glue
ZnO
Conventional TEM
preparation with final
Ar+ ion milling (5kV)
6P
ZnO
Ultramicrotomy preserves the crystalline structure of 6P!
Cross section TEM characterization
of organic/semiconductor hybrid structures
Left: ZO#1068a_UMC_cs4_t100nm, mesh B; hrtem02a_...._100kx_slit10eV_defocus-1um_..._c.jpg,
right : ZO#1068a_cs1, hrtem04_ZnO_6P_ZnO_ZA100_spot3_alpha1_CLAp2_HCAp0_300kx_infocus_c.tif,
Humboldt-Universität zu Berlin, Institut für Physik,
AG TEM und AG Photonik
Electropolishing
Conductive specimen (area 1 cm2)
lacquered around the edges acting
as the anode of an electrolytic cell
Progress of thinning: initial perforation
at the top region, covering, turning by
180°, perforation at the top, ...
final thinning at the center of the
sheet, electron transparent regions at
jagged edges, formatting, placing on a
grid
Chemical thinning
1. Chemical polishing using chemical solvent.
2. Removal of amorphized FIBsurfaces by dipping into, e.g. KOH.
Particles
Strategies of TEM preparation
DEPOSIT
evaporate
sputter
BULK
>3 mm
SURFACE/
INTERFACE
>3 mm
SMALL PIECES
>3 mm
PLAN
protect on
one side,
shield
lacquer
CROSS-SECTION
build up to
1.5 mm depth,
plate deposit embed,
glue face to face
FILM/
GRID
INITIAL
PREP.
cut,
lap,
grind
D
I
M
P
L
E
JET
electropol.
chem-pol.
ion mill
DISK
SHEET
cut
HAND
electropol.
chem-pol.
FOIL/
GRID
BLOCK
cut
ULTRA
MICROT.
SECTION/
GRID
ETCH
acid
REPLICATE
+ release etch?
EXTRACTION
REPLICA/
GRID
DISK
cut, punch, drill
M
I
R
O
S
C
O
P
CRUSH
pestle,
mortar
CLEAVE
razor,
blade
C
E
FRAGMENTS
SUPPORT FILM/
GRID
TEM preparation of small particles (1)
e- transparent particles (t < 100 nm)
• Dispersion in a non dissolving
liquid (e.g.: methanol, water, etc.)
in an ultrasonic bath
• Transfer to a carbon film
supported by a copper grid
Evaporation of a droplet
Dipping
TEM grids
and
many
more
holey carbon film
lacey carbon film
TEM preparation of small particles (1)
(NH4)2[(VO)3(P2O7)2] (AVP) powder on
Cu/C supporting grid seen through optical
microscope (cell width of the grid: 20 µm)
TEM bright-field image of Pd particles
on CaF2 support
TEM preparation of small particles (2)
Non-transparent particles
(t > 0.5 µm)
Particles distributed in solutions
Preparation for cryogenic TEM
Why vitrification of samples?
1. Preservation of the native state under high vacuum conditions
• Cryo-protection from high vacuum artifacts (like evaporation)
• Cryo-protection of organic structures against suffering
severe radiation damage since bonding energy << electron energy
• Cooling of the sample leads to reduced mobility of reactive species
2. Applications:
• Life science (viruses, cells, proteins, etc.)
• Materials science (self assembly in solution)
Preparation for cryogenic TEM
Instrumentation:
Plunge freezer
FEI Vitrobot
High-tilt
cryo transfer
holder
(Gatan)
JEOL JEM
2100Cryo
Preparation for cryogenic TEM
Vitrification of water
Cryofixation: Fast cooling rates (10000 K/s)
needed to inhibit crystallization of water
 Sample must be kept below -130°C
to avoid recrystallization
Hex ice
Cubic ice
amorphous
Preparation for cryogenic TEM
Cryogens:
Liquid ethane (C2H6):
Liquid N2:
- Fast cooling rate (thermal
conductivity 300!!! x higher than
LN2)
- Cooling rate higher than 105 K/s
needed to obtain vitrified water
- Application for vitrification of
water-based samples
- Poor heat conductance due to
Leidenfrost effect
- low difference in melting and
boiling point
- Application for vitrification of
tetrahydrofuran (THF) and
hexane-based samples
Leidenfrost effect
Suitable support film:
Lacey carbon film
Holey carbon film
Preparation for cryogenic TEM
Plunge freezing:
Preparation for cryogenic TEM
Cryo negative staining for contrast enhancement:
floating drop method
Established stains:
ammonium molybdate, uranyl acetate,
sodium tungstate, osmium tetroxide
Preparation for cryogenic TEM
Commercial plunge freezers:
Leica EM GP
FEI Vitrobot Mark IV
Gatan Cryoplunge 3
Preparation for cryogenic TEM
Examples of successfully prepared samples in vitrified water:
micellar structures
block-co-polymer micelles
(nano-flowers)
Conclusion
Strategies of TEM preparation
DEPOSIT
evaporate
sputter
BULK
>3 mm
SURFACE/
INTERFACE
>3 mm
SMALL PIECES
>3 mm
PLAN
protect on
one side,
shield
lacquer
CROSS-SECTION
build up to
1.5 mm depth,
plate deposit embed,
glue face to face
FILM/
GRID
INITIAL
PREP.
cut,
lap,
grind
D
I
M
P
L
E
JET
electropol.
chem-pol.
ion mill
DISK
SHEET
cut
HAND
electropol.
chem-pol.
FOIL/
GRID
BLOCK
cut
ULTRA
MICROT.
SECTION/
GRID
ETCH
acid
REPLICATE
+ release etch?
EXTRACTION
REPLICA/
GRID
DISK
cut, punch, drill
M
I
R
O
S
C
O
P
CRUSH
pestle,
mortar
CLEAVE
razor,
blade
C
E
FRAGMENTS
SUPPORT FILM/
GRID
References
Transmission Electron Microscopy, A textbook for materials science
D.B. Williams, C.B. Carter
Plenum Press New York 1996
Handbook of Microscopy,
Applications in Materials Science, Solid-State Physics and Chemistry
Ed. S. Amelincks, D. van Dyck, J. van Landuyt, G. van Tendeloo
VCH Verlagsgesellschaft Weinheim 1997
Specimen Preparation for Transmission Electron Microscopy of Materials
Microscopy Handbook 03
P.J.Goodhew
Oxford University Press, Royal Microscopical Society, 1984
Sample Preparation Handbook for Transmission Electron Microscopy
J. Ayache, L. Beaunier, J. Boumendil, G. Ehret, D. Laub
Springer 2010