Advanced SEM: ESEM and Cryo-SEM

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1 Advanced SEM: ESEM and Cryo-SEM Peter Harris

2 Electron Microscopy Scanning electron microscope Transmission electron microscope Electron gun Condenser lens Objective lens Specimen Projector lens Specimen Fluorescent screen Digital camera Wasp eye Adenovirus negatively stained with uranyl acetate

3 Conventional SEM High vacuum (~ 10-6 Torr) Dry samples Conductive samples Specimen 3

4 Water Many interesting samples contain water: Plant/animal tissues Food samples Gels/colloids Suspensions of particles 4

5 Air drying a hydrated sample 5

6 Drying Damage Leaf cross-section 6

7 Solutions to the water problem - 1 Drying solutions: Freeze Drying Critical Point Drying Workshop 14 November

8 Solutions to the water problem - 2 Non-drying solutions: ESEM: Cool sample & introduce water vapour into chamber Cryo-SEM: Freeze sample in liquid nitrogen and image in frozen state

9 History of ESEM Late 1970s: Development of ESEM by D. Danilatos (Australian Wool Corporation) Innovations Differentially pumped apertures for pressure separation. Cooled specimen to retain moisture. New (backscattered) electron detector. 1980s: ElectroScan ESEM. First successful commercial ESEM. Gaseous secondary electron detector. Today: FEI Company are at the forefront of ESEM development. Ref: V. Robinson, The development of variable pressure scanning electron microscopy, Microscopy and Analysis, Nov/Dec 2016, p.17.

10 ESEM: how it works Differential Pumping Pressure limiting aperture: typical chamber pressure 10 Torr Gaseous secondary electron detector Sample cooling: typical temperature 5 C Cooled specimen 10

11 ESEM samples don t have to be coated! Secondary electrons from sample strike water molecules. Positively charged water molecules are attracted to negatively charged sample. Negative charge at sample surface is neutralized. 11

12 ESEM plant specimens Cocoa embryo torpedo stage Basil leaf 12

13 ESEM plant specimens Raspberry buds : Hiroyuki Imanishi, Plant Sciences

14 ESEM plant specimens Drosera adelae stigma, ESEM

15 ESEM plant specimens Wild strawberry flower in early stages of growth - Rashed Alzahrani, SBS.

16 ESEM food specimens Potato, starch grains in situ Potato, starch grains washed out 16

17 ESEM: other samples Fat crystals Preserved nematode 17

18 ESEM: imaging water Condensation on ostrich feathers 18

19 ESEM: limitations Limited Resolution ~10x worse than conventional SEM Limited field of view Lack of contrast 19

20 Cryo-SEM: how it works 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 20

21 Cryo-SEM: how it works SEM mounted preparation chamber Quorum: PolarPrep

22 Cryo-SEM: how it works 22

23 Cryo-SEM: how it works 23

24 Cryo-SEM: how it works Preparation chamber: Fracturing/sublimation/coating

25 Cryo-SEM: how it works

26 Cryo-SEM: fracturing liquid samples Xanthan Gum in water Natural Greek yogurt 26

27 Cryo-SEM: freeze fracturing Cotoneaster leaf fractured at -140 C Grass leaf fractured at -190 C 27

28 Cryo-SEM: freeze fracturing Short dough biscuit - Dr Veronica Giacintucci FNS. Green areas are starch particles.

29 Cryo-SEM: sublimation Ice cream (-90 C for 5mins@ ~10-3 Pa) Ice cream (-90 C for ~10-3 Pa) 29

30 Cryo-SEM Cappuccino foam 60% oil in water emulsion 30

31 Cryo-SEM & ESEM: Summary Both: hydrated samples ESEM: less sample preparation ESEM: faster Cryo-SEM: higher magnifications Cryo-SEM: liquid samples 31

32 Becoming an EMLab user Sherrie Foo

33 Remaining workshops Date Topic 14 November November November 2017 Biological specimen preparation for SEM X-ray Analysis and Elemental Mapping Biological specimen preparation for TEM 33

34 Presentations available on website

35 Electron Microscopy Laboratory Questions? 35