esem and cryo-sem - university of reading · 12 esem and cryo-sem esem • change the microscope to...
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www.reading.ac.uk/cfam © University of Reading 2007 November 21, 2013
ESEM and Cryo-SEM Matthew Spink
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ESEM and Cryo-SEM 2
Overview
• Recap SEM
• Water and SEM
• Environmental SEM
• Cryo-SEM
• Comparison
• Questions/Demo
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ESEM and Cryo-SEM
SEM - Imaging the surface of samples
3
SEM is used to gain information about a sample’s topology and composition.
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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
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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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ESEM and Cryo-SEM 6
Conventional SEM
• Electron beam
• High vacuum
– ~10-4 Pa (~10-6 torr)
• Dry samples
• Conductive samples
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ESEM and Cryo-SEM 7
Water
• Many interesting samples contain water:
– Food samples
– Marine sediments
– Plant/animal tissues
– Gels/colloids
– Suspensions of particles
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ESEM and Cryo-SEM 8
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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ESEM and Cryo-SEM 9
Air drying a hydrated sample
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ESEM and Cryo-SEM 10
Drying Damage
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ESEM and Cryo-SEM 11
Solutions to the water problem
• Drying solutions:
– Freeze Drying
– Critical Point Drying
• Non-drying solutions:
– ESEM
– Cryo-SEM
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ESEM and Cryo-SEM 12
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 and Cryo-SEM 13
ESEM: Control of humidity
Humidity can be controlled by temperature
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ESEM and Cryo-SEM 14
ESEM: Control of humidity
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ESEM and Cryo-SEM 15
ESEM: control of chamber humidity
Condensation on ostrich feathers
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ESEM and Cryo-SEM 16
Pre
ssu
re
Temperature
Ice (solid)
Water Vapour (gas)
Water (liquid)
ESEM: phase diagram of water
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ESEM and Cryo-SEM 17
ESEM: components
Cooled specimen
• Differential Pumping
• Pressure limiting aperture
• Gaseous secondary electron
detector
• Sample cooling
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ESEM and Cryo-SEM
Cascade of secondary electrons in ESEM
• Primary electron beam
impinges on the sample
causing production of
backscattered and secondary
electrons
18
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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.
19
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ESEM and Cryo-SEM 20
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 and Cryo-SEM 21
ESEM: no sample prep
Cocoa embryo torpedo stage Basil leaf
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ESEM and Cryo-SEM 22
ESEM: minimal sample prep
Potato, starch grains in situ Potato, starch grains washed out
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ESEM and Cryo-SEM 23
ESEM: other samples
Fat crystals Preserved nematode
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ESEM and Cryo-SEM 24
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 and Cryo-SEM 25
ESEM: limitations
• Limited Resolution ~10x
worse than conventional SEM
• Slightly limited field of view
• Lack of contrast
Hemp leaf surface
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ESEM and Cryo-SEM 26
Cryo-SEM
• Change the sample to suit the
microscope
• Perform SEM on frozen
samples
Arabidopsis seed
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ESEM and Cryo-SEM 27
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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ESEM and Cryo-SEM 28
Pre
ssu
re
Temperature
Ice (solid)
Water Vapour (gas)
Water (liquid)
Cryo-SEM: phase diagram of water
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ESEM and Cryo-SEM 29
Cryo-SEM: fracturing liquid samples
Xanthan Gum in water Natural Greek yogurt
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ESEM and Cryo-SEM 30
Cryo-SEM: fracturing temperature
Cotoneaster leaf fractured at -140°C Grass leaf fractured at -190°C
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ESEM and Cryo-SEM 31
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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ESEM and Cryo-SEM 32
Cryo-SEM: FEI Quanta 600F
• SEM mounted preparation
chamber
• Quorum: PolarPrep 2000
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ESEM and Cryo-SEM 33
Cryo-SEM: procedure
4
3
2 1
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ESEM and Cryo-SEM 34
ESEM and cryo-SEM: common samples
Drosera adelae stigma, ESEM Drosera adelae stigma, cryo-SEM
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ESEM and Cryo-SEM 35
Cryo-SEM: unique samples
60% oil in water emulsion Cappuccino foam
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ESEM and Cryo-SEM 36
Cryo-SEM: magnification range
Natural Greek yoghurt Drosera adelae flower
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ESEM and Cryo-SEM 37
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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ESEM and Cryo-SEM 38
Demo/Questions?