advanced sem: esem and cryo-sem · em workshops 2017 • differentially pumped apertures for...
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www.reading.ac.uk/EMLab
Peter Harris
Advanced SEM: ESEM and Cryo-SEM
EM Workshops 2017
Electron Microscopy
Specimen
Condenser lens
Objective lens
Specimen
Projector lens
Fluorescent screen
Electron gun
Digital camera
Transmission electron microscopeScanning electron microscope
Adenovirus negatively stained with uranyl acetate
Wasp eye
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• High vacuum (~ 10-6 Torr)
• Dry samples
• Conductive samples
Specimen
Conventional SEM
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• Many interesting samples contain water:
– Plant/animal tissues
– Food samples
– Gels/colloids
– Suspensions of particles
Water
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Air drying a hydrated sample
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Leaf cross-section
Drying Damage
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• Drying solutions:
– Freeze Drying
– Critical Point Drying
Workshop 14 November
Solutions to the water problem - 1
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Non-drying solutions:
ESEM:Cool sample & introducewater vapour into chamber
Cryo-SEM:Freeze sample in liquid nitrogen and imagein frozen state
Solutions to the water problem - 2
EM Workshops 2017
• Differentially pumped apertures for pressure separation.
Innovations
1980s: ElectroScan ESEM. First successful commercial ESEM. Gaseous secondary electron detector.
• New (backscattered) electron detector.
Late 1970s: Development of ESEM by D. Danilatos (Australian Wool Corporation)
• Cooled specimen to retain moisture.
Today: FEI Company are at the forefront of ESEM development.
History of ESEM
Ref: V. Robinson, “The development of variable pressure scanning
electron microscopy”, Microscopy and Analysis, Nov/Dec 2016, p.17.
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ESEM: how it works
• Differential Pumping
• Pressure limiting aperture: typical
chamber pressure 10 Torr
• Gaseous secondary electron detector
• Sample cooling: typical temperature
5°CCooled specimen
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Secondary electronsfrom sample strike watermolecules.
Positively charged watermolecules are attracted tonegatively charged sample.
Negative charge at samplesurface is neutralized.
ESEM samples don’t have to be coated!
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ESEM – plant specimens
Cocoa embryo torpedo stage Basil leaf
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• Raspberry buds : Hiroyuki Imanishi, Plant Sciences
ESEM – plant specimens
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ESEM – plant specimens
Drosera adelae stigma, ESEM
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ESEM – plant specimens
Wild strawberry flower in early stages of growth - Rashed Alzahrani, SBS.
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Potato, starch grains in situ Potato, starch grains washed out
ESEM – food specimens
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ESEM: other samples
Fat crystals Preserved nematode
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ESEM: imaging water
Condensation on ostrich feathers
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ESEM: limitations
• Limited Resolution ~10x worse
than conventional SEM
• Limited field of view
• Lack of contrast
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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
Cryo-SEM: how it works
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• SEM mounted preparation
chamber
• Quorum: PolarPrep 2000
Cryo-SEM: how it works
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Cryo-SEM: how it works
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Cryo-SEM: how it works
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Preparation chamber:Fracturing/sublimation/coating
Cryo-SEM: how it works
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Cryo-SEM: how it works
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Cryo-SEM: fracturing liquid samples
Xanthan Gum in water Natural Greek yogurt
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Cryo-SEM: freeze fracturing
Cotoneaster leaf fractured at -140°C Grass leaf fractured at -190°C
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Cryo-SEM: freeze fracturing
Short dough biscuit - Dr Veronica Giacintucci FNS. Green areas are starch particles.
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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
60% oil in water emulsionCappuccino foam
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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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Becoming an EMLab user
Sherrie Foo
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Remaining workshops
Date Topic
14 November 2017Biological specimen
preparation for SEM
21 November 2017X-ray Analysis and
Elemental Mapping
28 November 2017Biological specimen
preparation for TEM
EM Workshops 2017
Presentations available on website
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► Questions?
Electron Microscopy Laboratory