Probe formation and basic image formation in SEM Using JEOL 5310LV as example s/esem-manual.swf.
ESEM and Cryo-SEM - University of Reading · 12 ESEM and Cryo-SEM ESEM • Change the microscope to...
Transcript of ESEM and Cryo-SEM - University of Reading · 12 ESEM and Cryo-SEM ESEM • Change the microscope to...
www.reading.ac.uk/cfam © University of Reading 2007 November 21, 2013
ESEM and Cryo-SEM Matthew Spink
ESEM and Cryo-SEM 2
Overview
• Recap SEM
• Water and SEM
• Environmental SEM
• Cryo-SEM
• Comparison
• Questions/Demo
ESEM and Cryo-SEM
SEM - Imaging the surface of samples
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SEM is used to gain information about a sample’s topology and composition.
ESEM and Cryo-SEM
• High energy “primary”
electrons hit sample surface
• “Secondary” electrons are
ejected from sample atoms
• Primary electron pass
through, into or are reflected
from sample
Primary beam 0.5-30kV
Backscatter electrons
Secondary electrons
SEM Recap
ESEM and Cryo-SEM
SEM Recap
•Primary beam is focused to a fine point •Then scanned across sample surface •Secondary electrons are emitted from specimen as the beam passes over it •Secondary electrons are detected and a picture is built up.
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Conventional SEM
• Electron beam
• High vacuum
– ~10-4 Pa (~10-6 torr)
• Dry samples
• Conductive samples
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Water
• Many interesting samples contain water:
– Food samples
– Marine sediments
– Plant/animal tissues
– Gels/colloids
– Suspensions of particles
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Phase diagram of water
0.01°C
611.7 Pa (4.6 torr)
100°C
98066.5 Pa (735.6 torr)
Pre
ssu
re
Temperature
Ice (solid)
Water Vapour (gas)
Water (liquid)
Conventional SEM
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Air drying a hydrated sample
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Drying Damage
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Solutions to the water problem
• Drying solutions:
– Freeze Drying
– Critical Point Drying
• Non-drying solutions:
– ESEM
– Cryo-SEM
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ESEM
• Change the microscope to suit the sample
• Preserving liquid water within the SEM chamber
• Sample cooling
• Introducing water vapour
Condensation
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ESEM: Control of humidity
Humidity can be controlled by temperature
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ESEM: Control of humidity
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ESEM: control of chamber humidity
Condensation on ostrich feathers
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Pre
ssu
re
Temperature
Ice (solid)
Water Vapour (gas)
Water (liquid)
ESEM: phase diagram of water
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ESEM: components
Cooled specimen
• Differential Pumping
• Pressure limiting aperture
• Gaseous secondary electron
detector
• Sample cooling
ESEM and Cryo-SEM
Cascade of secondary electrons in ESEM
• Primary electron beam
impinges on the sample
causing production of
backscattered and secondary
electrons
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ESEM and Cryo-SEM
Cascade of secondary electrons in ESEM
• Secondary electrons are
accelerated towards the
anode.
• Secondary electrons collide
with water vapour molecules
producing more secondary
electrons.
• This produces a cascade of
electrons which is detected.
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Secondary electrons from sample strike water molecules.
Positively charged water molecules are attracted to negatively charged sample.
Negative charge at sample surface is neutralized.
Adding water vapour in ESEM has the added benefit of negating the samples charge allowing non-conductive samples to be imaged.
Added benefit of water vapour in ESEM
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ESEM: no sample prep
Cocoa embryo torpedo stage Basil leaf
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ESEM: minimal sample prep
Potato, starch grains in situ Potato, starch grains washed out
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ESEM: other samples
Fat crystals Preserved nematode
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Gaseous secondary electron detector – fits on objective lens.
ESEM: FEI Quanta 600F
• Full ESEM capability
• Pressures up to 2666Pa / 20 torr
• Samples typically cooled to ~5°C
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ESEM: limitations
• Limited Resolution ~10x
worse than conventional SEM
• Slightly limited field of view
• Lack of contrast
Hemp leaf surface
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Cryo-SEM
• Change the sample to suit the
microscope
• Perform SEM on frozen
samples
Arabidopsis seed
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Cryo-SEM: how does it work?
1. Sample is frozen
2. Frozen sample is handled
under vacuum
3. Fractured/sublimation/coating
4. Frozen sample transferred to
SEM cryo stage
5. Normal high vacuum imaging
Cryo-stage
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Pre
ssu
re
Temperature
Ice (solid)
Water Vapour (gas)
Water (liquid)
Cryo-SEM: phase diagram of water
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Cryo-SEM: fracturing liquid samples
Xanthan Gum in water Natural Greek yogurt
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Cryo-SEM: fracturing temperature
Cotoneaster leaf fractured at -140°C Grass leaf fractured at -190°C
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Cryo-SEM: sublimation
Ice cream (-90°C for 15mins @ ~10-3 Pa) Ice cream (-90°C for 5mins@ ~10-3 Pa)
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Cryo-SEM: FEI Quanta 600F
• SEM mounted preparation
chamber
• Quorum: PolarPrep 2000
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Cryo-SEM: procedure
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ESEM and cryo-SEM: common samples
Drosera adelae stigma, ESEM Drosera adelae stigma, cryo-SEM
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Cryo-SEM: unique samples
60% oil in water emulsion Cappuccino foam
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Cryo-SEM: magnification range
Natural Greek yoghurt Drosera adelae flower
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Cryo-SEM & ESEM: Summary
• Both: hydrated samples
• ESEM: less sample preparation
• ESEM: faster
• Cryo-SEM: higher magnifications
• Cryo-SEM: liquid samples
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Demo/Questions?