Scanning electron microscopy- principles and applications

SEM

1. SCANNING ELECTRON MICROSCOPY Principles and Applications Vedang S. Junagade Reg.No.- PA/2023/107 Subject code – GE611 1

2. Flow of Contents 2

3. Secondary Electrons(SE) Inelastic scattering IonizationRemoval of Loosely bound e - SE are low energy e - with energy < 50eV Present superficially on the interaction zone Generally provide topographical information 1)Zhou , Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40 . 2) https://www.thermofisher.com/blog/materials/sem-signal-types-electrons-and-the-information-they-provide/ 3 SE detector image of leaf SE Generation Interaction zone

4. Back Scattered Electrons(BSE) Elastic scattering amongst specimen e - and e - beam BSE have e - with energy > 5eV Are present in depth at the ionization or interaction zone Sensitive to Atomic no. – More Atomic no, brighter the image Mostly used for Depth Analysis 1)Lam , Matilynn , et al. "An Introduction to Scanning Electron Microscopy and Science Communication Skills for Undergraduate Chemistry Students."  Journal of Chemical Education  100.7 (2023): 2802-2808 . 2)https ://www.thermofisher.com/blog/materials/sem-signal-types-electrons-and-the-information-they-provide/ 4 BSE generation BSE use case for different Atomic No.

5. SEM- An Overview SEM - utilizes SE produced by the interaction of the sample and the e - beam to yield topographical information Magnification – 300000 times In SEM a high energy e - bean is focused onto a specimen where SE are produced that show micromeritics, topography of the specimen SEM Model- FEI Quanta 250 FEG Abdullah, Avin , and Azad Mohammed. "Scanning electron microscopy (SEM): A review."  Proceedings of 2018 International Conference on Hydraulics and Pneumatics-HERVEX . Băile Govora Romania, 2019. 5 SEM pictorial image

6. SEM Components- Electron Gun Produces an e - beam by heat/extraction voltage for e - emission Is of 3 types- a) W filament b)LaB 6 Filament c)Field Emission Guns The cathode generates e - and the anode accelerates it by voltage application 1)Lam, Matilynn , et al. "An Introduction to Scanning Electron Microscopy and Science Communication Skills for Undergraduate Chemistry Students."  Journal of Chemical Education  100.7 (2023): 2802-2808 . 2)https ://myscope.training/pdf/MyScope_SEM.pdf 6 Types of Electron Guns Electron Gun

7. Electromagnetic Lenses A pair of electromagnets that narrow down/ converges the e - beam onto the specimen A metallic pole + wires coiled around itself to produce B o 2 pairs of lenses used- a) Condenser b) Objective A condenser e - beam converges and made uniform 2 pole pieces upon which Cu wire wounded up to generate B o An Objective Focuses e - beam on the specimen probe Zhou , Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40. 7 Electromagnetic Lenses Narrowing down of the beam

8. Scanning Coils Deflect the e - beam , and scan the specimen in XY axis 2 pairs of electromagnetic coils which deflect the beam/moves the beam in a point to point fashion to create a rectangular raster They deflect the beam horizontally and vertically onto the specimen – Rastering 1)Zhou, Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40. 2) https://myscope.training/pdf/MyScope_SEM.pdf 8 Scan Coils Representation of rastering

9. Detection Systems The detector for SE - Everhart-Thornley detector/ET detector Comprises of 3 components The scintillator - converts the coming SE/ electrical signal to light/photons The light pipe – Passes the photons to the PMT and due to its position ensures maximum collection of SE PMT- The photomultiplier tube converts the photons/light to Electrical signal The electrical signal produced then gets sent for imaging to obtain the topographical image 1)Lam, Matilynn , et al. "An Introduction to Scanning Electron Microscopy and Science Communication Skills for Undergraduate Chemistry Students."  Journal of Chemical Education  100.7 (2023): 2802-2808 . 2)https ://myscope.training/pdf/MyScope_SEM.pdf 9 Layout of ET detector BSE detector

10. Vacuum Systems Indispensable part of SEM , prevents unwanted scattering of e - and other contamination 2 commonly used pumps- a)Mechanical Pump b) Diffusion Pumps Mechanical Pump A motor driven rotor that compresses High vol. of gas and increases the overall compressed gas pressure When the compressed gas pressure reaches a certain threshold it gets expelled out Vacuum achieved- 5 x 10 -5 Torr Zhou , Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40. 10

11. Diffusion Pumps A vaporized oil circulates from top to bottom The gases present goes to the top carried away by the vaporized oil The gases gets discharged and the vaporized oil gets cooled and recirculated Vacuum achieved - 5 x 10 -5 Torr 11

12. Applications –Biological Sciences SEM Colorized image of sperm tails tangled up in seminiferous tubules E-SEM Colorized image of artery with RBCs Wide area SEM image of murine kidney https://assets.thermofisher.com/TFS-Assets/MSD/brochures/life-sciences-sem-brochure-br0179.pdf 12

13. Applications – Geology SEM image of Anthophyllite Asbestos SEM image of Winchite-Richterite Asbestos SEM image of Tremolite Asbestos SEM image of a mineral where Bright portions indicate high atomic no elements and dark indicate low atomic no elements https://www.usgs.gov/media/galleries/fibrous-and-asbestiform-minerals 13

14. Applications- Pharmaceuticals SEM micrograph of Triamcinolone acetanide SEM micrograph of Betamethasone Valerate SEM micrograph of raw Beclomethasone Propionate SEM m icrograph of Processed Beclomethasone Propionate Rasenack , Norbert, Hartwig Steckel , and Bernd W. Müller. " Micronization of anti-inflammatory drugs for pulmonary delivery by a controlled crystallization process."  Journal of pharmaceutical sciences  92.1 (2003): 35-44. 14

15. Factors affecting Image Quality – Accelerating Voltage If voltage gets increased- Increased penetration and interaction volume Can lead to high BSE generation Typical Voltage range – 5-30 KV Low voltage – SE- Surface observation and topography High voltage – BSE – Depth analysis 15 SEM image of CaF 2 at 1kV SEM image of CaF 2 at 20 kV Zhou , Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40.

16. Spot Size Size/Cross Section of the beam made on the specimen It affects the a)Resolution b) No of e - generated c) Graininess of the image When the spot size is more a blur image will be generated and will have poor resolution A smaller spot size produces a clear image and has a good resolution respectively. 1)Zhou , Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40 . 2)https ://myscope.training/pdf/MyScope_SEM.pdf 16 Diatom image Smaller spot size Diatom image Larger Spot Size

17. Working distance and Depth of field Working distance – Distance between the bottom of the SEM column and the top of the specimen A smaller working distance – may produce high resolution image(10mm or small) A larger working distance – Low resolution image Depth of Field – Zone where the specimen image appears to be focused on the eye This can be seen by altering or slightly increasing the working distance(the beam converging angle decreases ) 1) Zhou, Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40 . 2)https :// myscope.training/pdf/MyScope_SEM.pdf 17 SEM image showing depth of field

18. 18 ZnO nanowires Image with working distance – 3mm ZnO nanowires Image with working distance – 12mm Zhou , Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40.

19. Aperture Size A minute hole placed in a strip of metal placed in the path of e - beam to restrict or limit the e - beam and e - that are off-axis Can also narrow the beam below the aperture depending upon the aperture selected Objective lens aperture – reduces/ excludes extraneous e - Large aperture- Low magnification Small Aperture – High magnification Zhou , Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40. 19 ZnO Image with larger aperture size ZnO Image with smaller aperture size

20. Magnification Magnification refers to enlargement of image / a particular portion of the image In SEM achieved by scanning a smaller area A scale bar present on the image- Gives the unit of distance on the image Magnification adjusted by the scanning coils 20 Zhou, Weilie , et al. "Fundamentals of scanning electron microscopy (SEM)."  Scanning microscopy for nanotechnology: techniques and applications  (2007): 1-40.

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22. Specimen Preparation M aterials that has variable sample prep. – a)Desiccated and conductive b)Desiccated and non-conductive c)Wet samples a)For conductive specimens- We keep the specimen secured onto the pin stub with the help of C tape and visualize b)For nonconductive specimens – We coat the specimen with a conductive layer (Au ) that protects the specimen from overheating and gives good image. A sputter coater used where Ar gas used to knock Au atoms off the Au coil so they coat the specimen surface c)Wet samples- A technique called ESEM gets used where a low temperature gets used at high vacuum condition Deben Coolstage used to visualize specimens at -25 o C Nguyen, Jenny Ngoc Tran, and Amanda M. Harbison. "Scanning electron microscopy sample preparation and imaging."  Molecular Profiling: Methods and Protocols  (2017): 71-84. 22 S pecimen probe Sample Coating

23. Application- Case Study Spherical crystallization was done by mixing Acetone+PVP in distilled water and added to mech. stirrer to get spherical aggregates. Bridging liquid CHCl 3 was added followed by filtration and drying at 24hrs at RT. Gupta, Venkadari Rammohan , et al. "Spherical crystals of celecoxib to improve solubility, dissolution rate and micromeritic properties."  Acta pharmaceutica  57.2 (2007): 173-184. 23

24. SEM image before crystallization Pure Celecoxib SEM image after treatment Of spherical crystallized Celecoxib 24

25. THANK YOU 25