The document presents an overview of scanning electron microscopy (SEM), detailing the generation of secondary and back-scattered electrons, and the components involved, including electron guns, electromagnetic lenses, and detection systems. It outlines key applications in biological sciences, geology, and pharmaceuticals, as well as factors affecting image quality. Additionally, it discusses specimen preparation techniques to enhance imaging results.

1. SCANNING ELECTRON
MICROSCOPY
Principles and Applications
Vedang S. Junagade
Reg.No.- PA/2023/107
Subject code – GE611
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2. Flow of Contents
Generation of
Secondary Electrons
Generation of Back-
Scattered Electrons
Components of SEM-
Electron Gun
Components of SEM-
Detection system
Components of SEM-
Scan Coils
Components of SEM-
Electromagnetic
Lenses
Applications in
Biological Science,
Geology and
Pharmaceuticals
Factors affecting Image
Quality
Case Study
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)LaB6 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 Bo
• 2 pairs of lenses used- a) Condenser b) Objective
A condenser
o e- beam converges and made uniform
o 2 pole pieces upon which Cu wire wounded up to generate Bo
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
o A motor driven rotor that compresses High vol. of gas and increases the
overall compressed gas pressure
o When the compressed gas pressure reaches a certain threshold it gets
expelled out
o 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
o A vaporized oil circulates from top to bottom
o The gases present goes to the top carried away by the vaporized oil
o The gases gets discharged and the vaporized oil gets cooled and
recirculated
o Vacuum achieved - 5 x 10-5 Torr
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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
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14. Applications- Pharmaceuticals
SEM micrograph of
Triamcinolone acetanide
SEM micrograph of
Betamethasone Valerate
SEM micrograph of raw Beclomethasone
Propionate
SEM micrograph 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
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SEM image of CaF2 at 1kV SEM image of CaF2 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
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Zhou, Weilie, et al. "Fundamentals of scanning electron microscopy (SEM)." Scanning microscopy for nanotechnology: techniques and applications (2007): 1-40.

21. 21

22. Specimen Preparation
Materials that has variable sample prep. –
a)Desiccated and conductive b)Desiccated and non-conductive c)Wet samples
a)For conductive specimens-
o We keep the specimen secured onto the pin stub with the help of C tape
and visualize
b)For nonconductive specimens –
o We coat the specimen with a conductive layer (Au) that protects the
specimen from overheating and gives good image.
o 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-
o A technique called ESEM gets used where a low temperature gets used at
high vacuum condition
o Deben Coolstage used to visualize specimens at -25oC
Nguyen, Jenny Ngoc Tran, and Amanda M. Harbison. "Scanning electron microscopy sample preparation and imaging." Molecular
Profiling: Methods and Protocols (2017): 71-84.
22
Specimen 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 CHCl3 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.
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24. SEM image before crystallization
Pure Celecoxib
SEM image after treatment
Of spherical crystallized Celecoxib
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25. THANK YOU
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