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

4

3

2 1

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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?