Dr. Kim Rensing's Presentation Slides

Coating Techniques for Electron Microscopy
– Obtain the Best Images Possible
Speaker:
Dr. Kim Rensing, Applications Specialist, Leica Microsystems GmbH
Webinar Host:
Dr. Ami Miller, Life Science Editor of SelectScience
Coating Techniques for
Electron Microscopy
Kim Rensing PhD

Applications Specialist
Leica Microsystems
Overview Living up to Life
1. Specimen requirements
2. Coating quality
3. Coating techniques
4. Cryo-techniques
www.leica-microsystems.com
Specimen Requirements Living up to Life
Coating is always performed in vacuum
• Specimen must be suitable for vacuum - specimen vapour

pressure must be less than vacuum (i.e. cannot boil)
• Specimen must be solid - aqueous specimens must be

dehydrated, critical point dried or frozen.
Coating quality depends on: Living up to Life
• Coating technique
• Coating material
• Vacuum conditions
• Specimen temperature
• Layer thickness
• The specimen itself (“decoration effects”)
• Effects after coating
Coating Techniques Living up to Life
Sputtering:
• Metals
Evaporation:
• Carbon
• Metals
• Non-conductive materials
Living up to Life
Poll Question 1:
Which microscopy applications are your samples being

used for after coating?
- Light Microscopy
- SEM low magnification
- SEM high magnification
- TEM application
Carbon Coating
Carbon Coating Living up to Life
Properties
• Chemically inert, does not react with specimen or acids
• Stable in the electron beam
• Transparent for the electron beam (does not provide contrast!)
• Electrically conductive - reduces charging
Applications
• Carbon films or carbon films with holes for TEM
• Backing of collodion and formvar films for biological EM
• Conductive layers for EDX analysis
• Backing layers for replica techniques
Carbon thread evaporation
• Thermal evaporation in low or high vacuum

• Uniform, conductive films showing fine grains even
for corrugated surfaces
• Easy to handle
• Heat transfer:
• High with flashing
• Low with pulsing!!
Carbon Coating with Pulses Living up to Life
Very precise control of thickness using a quartz monitor!
ACE200
1.5 cm
Carbon Distribution at Low Vacuum Living up to Life
ACE200 20 nm Carbon on Mylar sheets
plus 20 mm
plus 10 mm
0 mm
Relative Density
0 1 2 3 4 5 6 7 8
Stage Diameter (cm)
Carbon Rod Evaporation (only in high vacuum)
• Thermal or sparking-arc evaporation

in high vacuum
• Uniform, conductive films showing
fine grains
• High heat transfer to the specimen
ACE600 • Poor control of coating rate
Electron Beam Evaporation: Carbon/Metal Living up to Life
 High vacuum only
 Very fine-grained layers:

• Carbon, Pt/C, W, Ta/W, Cr)
BAF060
 Directional beam produces
shadowing
 Low heat transfer to specimen
ACE600
Electron Beam Evaporation Living up to Life
• Electrons from tungsten filament are focused on source (anode) by electric field
-1300 V (Voltage depends on material)
e +
- Neutral
(evaporated)
atoms
e e-
-
+1300 V
Anode Tungsten
(Pt/C, Cr, W, C) filament
• Low heat transfer to the specimen

• Directional films showing very fine grains
• Facilitates layer thickness monitoring
Metal Coating
Reasons for Metal Coating Living up to Life
• Reduce charging
• Localize secondary electron (SE) and back-scattered

electron (BSE) signal to the surface in SEM
• Reduce heating of non-conductive specimens to

increase potential exposure time to the electron beam
• Reveal topography with shadowing (TEM or SEM)
Electron Beam Interaction Volume Living up to Life
Image Source: http://serc.carleton.edu/research_education/geochemsheets/electroninteractions.html
Electron Beam Interaction Volume Living up to Life
Uncoated Coated
Primary electron Primary electron

beam beam
Platinum
Formation of a Coating Layer in VacuumLiving up to Life
1. Condensation 2. Nucleation
condensation nucleation
grain growth coalescence
3. Grain growth 4. Coalescence
Coating Quality – Coating Thickness Living up to Life
Sputtered layers of different metals and different layer thicknesses
Pt 1 nm W 1 nm Cr 1 nm
20 nm 20 nm 20 nm
Starting pressure: 10-5 mbar; Argon pressure: 2X10-2 mbar; room temperature; TEM imaging
Coating Quality – Layer Thickness Living up to Life
Coating Quality – Layer Thickness Living up to Life
Layer thickness measurement (QSG 100)

Oscillating quartz:
• Indirect determination of layer thickness
• Frequency changes depend on density of coated material
• Layer thickness based on changes in the oscillating frequency
1.5 cm
ACE200 Low Vacuum Coater Living up to Life
Gold distribution across the stage at different heights.

Relative Density
Stage Diameter (cm)
ACE200 - Effect of Layer Thickness Living up to Life
10 nm gold 5 nm gold 2 nm gold
Living up to Life
Poll Question 2:
Which materials are you currently using for coating?
- Gold
- Platinum
- Gold/Palladium
- Platinum/Palladium
- Copper
- Iridium
- Chromium
- Carbon Rod
- Carbon Thread
ACE200– Effect of Material Living up to Life
5 nm gold 5 nm platinum
Metal Coating: Sputtering Living up to Life
Pre-vacuum (10-3 mbar) sputtering
 The vacuum before the sputter

process determines coating quality
 Coating materials: Au, Au/Pd, Pt
 Uniform coatings of samples sufficient

for tungsten, LaB6 SEMs
SCD 050
ACE200
Planar Magnetron Sputtering Living up to Life
-400…2000 V Vacuum chamber
Ar inlet
…Target atom
…e- Magnetic system
Ar+
…Ar+ ion Target: Cathode (-)
Ar+ Ar+
…Ar Ar+
Ar+ Ar+ Ar+ Ar+
Ar+
Ar+ Anode ring
Ar+
Ar plasma
Specimen
Deposited atoms
Specimen table
Pumping system
ACE200 Sputter Coated Platinum Living up to Life
5 nm Diffuse 5 nm Directional
Coating quality – Vacuum Conditions Living up to Life
• Quality depends mainly on the partial vapour

pressure in the chamber
• High vacuum before coating reduces vapour

pressure
Metal Coating: Sputtering Living up to Life
High vacuum (10-5 to 10-7 mbar) sputtering
 Magnetron Sputtering under high

(pre-sputtering) vacuum conditions
 Coating material: Conventional materials
(Au, Au/Pd, Pt) and Ir, W, Cr, Ta …
 Fine grained, uniform coatings
 Best for high-resolution FE-SEMs
ACE600
ACE600 - High Vacuum Coater Living up to Life
10 nm sputter coated gold
Coating Quality - Specimen TemperatureLiving up to Life
• Reorganization of the coating material depends

on the surface temperature.
• Colder temperature reduces mobility of the

coating material causing:
• Smaller distance between grains

• Finer structure
Coating Quality - Vacuum and Temperature Living up to Life
Platinum, Platinum, Platinum,

Low Vacuum High Vacuum High Vacuum,
Substrate at 25°C Substrate at 25°C Substrate at –80°C
Thinner coatings show

the surface structure.
Thicker coatings reflect

organization of the
coating material.
Mueller T., Walther P., Scheidegger C., Reichelt R., Mueller S. and Guggenheim R. (1990). Cryo-preparation and planar magnetron sputtering for
low temperature scanning electron microscopy. Scanning Microscopy 4 (4), 863-876.
Living up to Life
Poll Question 3:
How would you describe your sample topography?
- Rough sample surface

- Smooth sample surface
Coating Quality – Specimen Effects Living up to Life
• Physical and chemical differences in the

surface affect coating deposition
• Preferred condensation at specific surface

features create “decoration” effects
Coating Quality – Specimen Effects Living up to Life
PE - Primary electrons
SE - Secondary electrons
PE
R - Interaction volume
PE PE
PE
SE
F
R SE SE
R
R SE
R
R
Specimen
Coating Quality – Post Coating Effects Living up to Life
• Oxidation and reorganization produce aggregates
• Depends on temperature and humidity
• Causes contrast changes
Spider Mite Living up to Life
CRYO-SEM Techniques Living up to Life
High pressure freezing (for cells or tissues)
Freeze-fracturing or cryoplaning
Sublimation
Coating
Cryo-SEM
VCT100 – Vacuum Cryo Transfer Living up to Life
Cryo-Planing
Cryo-SEM
HPF Freeze Fracture,

Sublimation &
Coating
Cryo-SEM Living up to Life
Cryo-SEM
SE signal
Platinum
Frozen hydrated specimen
CryoSEM – Cryoplaned Poplar Wood Living up to Life
CryoSEM – Fractured Mouse Brain Living up to Life
Electron Microscopy Center Zurich (EMEZ)
Unidirectional Shadowing Living up to Life
Coating
Shadowing (~20 nm)
(~2nm)
Ice
Unidirectional Shadowing Living up to Life
BSE
Cryo-SEM
(Backscattered signal)
SE
Carbon
Platinum
Ice
Frozen hydrated biological specimen
Unidirectional Shadowing for CryoSEM Living up to Life
1µm
Elektronenmikroskopie ETH Zürich
Living up to Life
Thank you for attending
We hope you found the webinar useful and informative.
If you have any further questions please email

editor@selectscience.net.

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Coating Techniques for Electron Microscopy

– Obtain the Best Images Possible

Kim Rensing PhD

Coating is always performed in vacuum

• Specimen must be suitable for vacuum - specimen vapour

• Specimen must be solid - aqueous specimens must be

Which microscopy applications are your samples being

Carbon thread evaporation

• Thermal evaporation in low or high vacuum

Very precise control of thickness using a quartz monitor!

ACE200 20 nm Carbon on Mylar sheets

Carbon Rod Evaporation (only in high vacuum)

• Thermal or sparking-arc evaporation

 High vacuum only

 Very fine-grained layers:

 Low heat transfer to specimen

-1300 V (Voltage depends on material)

• Low heat transfer to the specimen

• Localize secondary electron (SE) and back-scattered

• Reduce heating of non-conductive specimens to

• Reveal topography with shadowing (TEM or SEM)

Image Source: http://serc.carleton.edu/research_education/geochemsheets/electroninteractions.html

Primary electron Primary electron

grain growth coalescence

3. Grain growth 4. Coalescence

Sputtered layers of different metals and different layer thicknesses

Layer thickness measurement (QSG 100)

Gold distribution across the stage at different heights.

Stage Diameter (cm)

10 nm gold 5 nm gold 2 nm gold

Pre-vacuum (10-3 mbar) sputtering

 The vacuum before the sputter

 Uniform coatings of samples sufficient

-400…2000 V Vacuum chamber

• Quality depends mainly on the partial vapour

• High vacuum before coating reduces vapour

High vacuum (10-5 to 10-7 mbar) sputtering

 Magnetron Sputtering under high

10 nm sputter coated gold

• Reorganization of the coating material depends

• Colder temperature reduces mobility of the

• Smaller distance between grains

Platinum, Platinum, Platinum,

Thinner coatings show

Thicker coatings reflect

How would you describe your sample topography?

- Rough sample surface

• Physical and chemical differences in the

• Preferred condensation at specific surface

• Oxidation and reorganization produce aggregates

• Depends on temperature and humidity

• Causes contrast changes

High pressure freezing (for cells or tissues)

HPF Freeze Fracture,

Electron Microscopy Center Zurich (EMEZ)

If you have any further questions please email

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