Scanning Electron Microscope (SEM)

2. Electron Microscope
Electron Microscopes are scientific instruments that use a
beam of highly energetic electrons to examine objects on a
very fine scale.
This examination can yield information about the:
• Topography
• Morphology
• Composition
• Crystallographic information

5. Mainly 2 types:
• Transmission Electron Microscope
(TEM) - allows one the study of the inner
structures.
• Scanning Electron Microscope (SEM) -
used to visualize the surface of objects.

6. Differences between SEM and TEM
TEM SEM
Electron beam passes through thin
sample.
Electron beam scans over surface of
sample.
Specially prepared thin samples are
supported on TEM grids.
Sample can be any thickness and is
mounted on an aluminum stub.
Specimen stage halfway down
column.
Specimen stage in the chamber at the
bottom of the column.
Image shown on fluorescent screen. Image shown on TV monitor.
Image is a two dimensional
projection of the sample.
Image is of the surface of the sample

7. Compound microscope image TEM image
Budding yeast cell
E. coli bacteria
Compound microscope image TEM image SEM image
SEM image

8. First Electron Microscope
Invented by Ernst Ruska
Year-1933
 He was awarded the Nobel
Prize for physics for his
invention in 1986

9. History of sem
 First SEM – debuted in 1938 by
Manfred Von Ardenne
 In 1965, Cambridge Scientific
Instrument (UK) & JOEL (Japan)
first commercialized SEM
individually.

11. SEM
 Produces images of a sample by
scanning it with a focused beam
of electrons in a raster scan pattern
 Electrons interact with atoms --
produces various signals that contain
information about the sample's
surface topography and composition.

15. 1.Electron cannon.
2. Electro-magnetic
lenses to focus the
electron beam .3. Vacuum pumps
system
.
4.Opening to insert the
object into the high-
vacuum observation
chamber.
5. Operation panel with
focus, alignment and
magnification tools and
a joystick for positioning
of the sample.
6. Screen for menu
and image display
7.Cryo-unit to prepare
frozen material before
insertion in the
observation chamber
in Cryo-SEM mode
PARTS OF SEM

16.  Electron gun consisting of
cathode and anode.
 The condenser lens
controls the amount of
electrons travelling down
the column
 The objective lens focuses
the beam into a spot on
the sample.
 Deflection coil helps to
deflect the electron beam.
 SED attracts the
secondary electrons.
 Additional sensors detect
backscattered electrons
and X-rays.

17. SEM SAMPLE PREPARATION
Appropriate size samples --- fit in the
specimen chamber
Mounted rigidly on a specimen holder--
specimen stub
aluminiumstubs

18. SEM SAMPLE PREPARATION
For imaging in the SEM, specimens must
be
 Electrically conductive
 Electrically grounded

19. SEM SAMPLE PREPARATION
1. Cleaning the surface of the specimen
2. Stabilizing the specimen
3. Rinsing the specimen
4. Dehydrating the specimen
5. Drying the specimen
6. Mounting the specimen
7. Coating the specimen

20. SEM SAMPLE PREPARATION
Cleaning the surface of the specimen
 Very important
 Surface contains many unwanted deposits,
such as dust, mud, soil etc

21. SEM SAMPLE PREPARATION
Stabilizing the specimen
 Hard, dry materials such as wood, bone, feathers,
dried insects, or shells can be examined with little
further treatment, but living cells and tissues and
whole, soft-bodied organisms usually require
chemical fixation to preserve and stabilize their
structure.
 Stabilization is typically done with fixatives.

22. SEM SAMPLE PREPARATION
 Fixation -- performed by incubation in a
solution of a buffered chemical fixative, such
as glutaraldehyde, sometimes in combination
with formaldehyde and other fixatives.
Fixatives that can be used are:-
1. Aldehydes.
2. Osmium tetroxide.
3. Tanic acid.
4. Thiocarbohydrazides.

23. SEM SAMPLE PREPARATION
Rinsing the specimen
 Sample must be rinsed -- remove excessive
fixatives.

24. SEM SAMPLE PREPARATION
Dehydrating the specimen
 Water must be removed
 Air-drying causes collapse and shrinkage, this
is commonly achieved by replacement
of water in the cells with organic solvents such
as ethanol or acetone.
 Dehydration -- performed with a graded
series of ethanol or acetone.

25. SEM SAMPLE PREPARATION
Drying the specimen
 Specimen should be completely dry
 Otherwise the sample will be destroyed

26. SEM SAMPLE PREPARATION
Mounting the specimen
 Specimen has to be mounted on the holder
 Mounted rigidly on a specimen holder called a
specimen stub
 Dry specimen -- mounted on a specimen stub
using an adhesive such as epoxy resin or
electrically conductive double-sided adhesive
tape.

27. Charge-up

29.  This charge-up phenomenon
can be prevented by coating
the non-conductor sample with
metal (conductor).

30. Sample coating is
intended to prevent
charge-up phenomenon
by allowing the charge
on the specimen surface
go to ground through
the coated conductive
film.

31. SEM SAMPLE PREPARATION
Coating the specimen
 To increase the conductivity of the
specimen and to prevent the high
voltage charge on the specimen
 Coated with thin layer i.e., 20nm-30nm
of conductive metal.
 All metals are conductive and require no
preparation before being used.

32. SEM SAMPLE PREPARATION
Coating the specimen
 Non-metals need to be made conductive
 Done by using a device called a "sputter
coater.”
Conductive materials
 Gold
 Gold-palladium Alloy
 Platinum
 Osmium
 Iridium
 Tungsten
 Chromium
 Graphite

33. “Sputter Coater”

34. A spider coated in gold

35. Electron beam-sample interactions
 The incident electron beam is scattered in the sample,
both elastically and inelastically
 This gives rise to various signals that we can detect
 Interaction volume increases with increasing
acceleration voltage and decreases with increasing
atomic number
Images: Smith College Northampton, Massachusetts

36.  So now we have a beam that is
scanning across the sample surface and
this beam is synched to the beam of
a CRT.

40. Detectors
Image: Anders W. B. Skilbred, UiO
Secondary electron detector
Backscattered electron
detector

42. • A secondary electron detector attracts the scattered electrons and,
depending on the number of electrons that reach the detector,
registers different levels of brightness on a monitor.

44.  A low atomic weight
area of the sample will
not emit as many
backscattered
electrons as a high
atomic weight area of
the sample.
 In reality, the image
is mapping out the
density of the sample
surface.

46. How do we get an image?
156 electrons!
Image
Detector
Electron gun
288 electrons!

47. SCANNING
ELECTRON
MICROSCOPIC
IMAGE OF THE
TONGUE

48. Scanning electron micrographs of the early human embryo

49.  This form of image processing is only in gray
scale which is why SEM images are always in
black and white.
 These images can be colorized through the use
of feature-detection software, or simply by hand
editing using a hand graphic editor.
 This is usually for aesthetic effects, for clarifying
structure, or for adding a realistic effect to the
sample.

50. Pollen and Stamens Wool fibers

51. So how does a SEM change the magnification of an image?
 By reducing the size of the area scanned
by the scan coils, the SEM changes the
magnification of the image.

52. Vacuum Chamber
 SEMs require a vacuum to operate.
 Without a vacuum, the electron beam generated by the
electron gun would encounter constant interference from
air particles in the atmosphere.
 Not only would these particles block the path of the
electron beam, they would also be knocked out of the air
and onto the specimen, which would distort the surface
of the specimen.

53. BIOLOGICAL APPLICATIONS OF
SEM
 Virology - for investigations of virus structure
 Cryo-electron microscopy – Images can be made of the
surface of frozen materials.
 3D tissue imaging -
– Helps to know how cells are organized in a 3D network
– Their organization determines how cells can interact.
 Forensics - SEM reveals the presence of materials on
evidences that is otherwise undetectable
 SEM renders detailed 3-D images
– extremely small microorganisms
– anatomical pictures of insect, worm, spore, or other
organic structures

54. SEM Advantages
 Gives detailed 3D and topographical imaging
and the versatile information garnered from
different detectors.
 Works very fast.
 Modern SEMs allow for the generation of
data in digital form.
 Most SEM samples require minimal
preparation actions.

55. SEM Disadvantages
 SEMs are expensive and large.
 Special training is required to operate an
SEM.
 Preparation of samples can result in
artifacts.
 Limited to solid samples.
 Carry a small risk of radiation exposure
associated with the electrons that scatter
from beneath the sample surface.

56. Thank you