ANOTHER PPT PRESENTATION FOR MY MEDICAL N PATHOLOGIST FRIENDS. THIS PPT WILL HELP U FOR SURE

1. P R E S E N T E D B Y : -
D R . G A U R A V S . S A L U N K H E
2 N D M D S
O R A L & M A X I L L O F A C I A L P A T H O L O G Y
SCANNING ELECTRON
MICROSCOPE

2. Contents
 Introduction
 History
 Construction of SEM
 Basics of specimen preparation
 Why images are visible

3. History
 The development of a SEM began a few yrs after the
invention of a TEM by Ruska in 1931, but the
commercialization of the SEM required about 30 yrs.
 In 1935, the original prototype of the SEM, which
scans the specimen with an e- beam to obtain an
image, was made by Knoll(Germany).
 In 1942, Zworykin (USA), developed a SEM for
observing a bulk specimens.
 In 1965, Cambridge Scientific Instrument
(UK) & JOEL (Japan) first commercialized
SEM individually.

4. Construction of SEM
A) Electron optical system.(to produce e-)
 electron gun, condenser lens, objective lens,
scanning coil.
B) Specimen stage (to place the specimen).
C) Secondary e- detector (to collect secondary e-).
D) Image display unit.
E) Operating system.
The electron optical system and a space surrounding
the specimen are kept at vacuum.

5. Construction of SEM
 Electron Gun- it produces an e- beam.
 It is a thermionic emission gun. (TEG), the thermo
electrons are emitted from a filament (cathode)
made of a thin tungsten wire, (about 0.1mm ) by
heating the filament at high temperature (about
2800k). Tungsten filament assembly

6. Construction of SEM
 These e- are gathered as an e-beam, flowing into the
metal plate (anode) by applying a positive voltage.
 If the hole is made at the center of the plate the e-
beam flows through this hole.
 If an electrode (Wehnelt electrode)is placed between
the cathode and anode and applied with the negative
charge, the speed of the e- beam can be adjusted.

7. Construction of SEM
 Condenser lens/Electromagnetic lens- placing a lens
below the e- gun enables to adjust the diameter of
the e- beam.
 A fine e- beam is requires for SEM .
A typical Electro Magnetic Lens

8.  Role of the condenser lens in formation of fine e-
beam
 The aperture is placed between the condenser lens
and objective lens.
 The aperture made of a thin metal plate, has a small
hole.
 The e- beam, which passed through the condenser
lens, illuminates this aperture-plate.
 The aperture allows a part of the e- beam to reach
the objective lens.

9. Construction of SEM
 If the excitation of the condenser lens is increased,
the e- beam greatly broadens on the aperture and
therefore the number of the electrons reaching the
objective lens decreases.
 To the contrary, if the excitation of the condenser
lens is decreased, the e- bean doesn’t broaden very
much and therefore, most of the electrons pass
through the aperture and many electrons reaches the
objective lens.
 In this way the e- probe diameter and the probe
current can be adjusted.

10. Construction of SEM
 Objective lens- the objective lens is used for focusing.

11. Construction of SEM
 Specimen stage- It supports the specimen.
 Moves smoothly.
 It can perform horizontal (x,y axis) & vertical
movements(z axis), tilting of specimen, and rotation.
 The horizontal movement is used for selection of the
field while the vertical movement is used to change
the image resolution.
SEM opened sample chamber

12.  Secondary e- detector- It is used for detecting the
secondary e- emitted from the specimen.
 A scintillator (fluorescent substance) is coated on the
tip of the detector and a high voltage of about 10 kV
is applied to it.
 The secondary e- are attracted to this high voltage
and then generate light when they hit the scintillator.
 This light is directed to a photon-multiplier tube
(PMT) through a light guide.
 The light is converted to the electrons, and these are
amplified as an electric signal.

13. Secondary electron
detector
Everhard -Thornley
Detector

14.  A supplementary electrode, called the collector, is
placed before the scintillator, and is applied with few
hundreds of voltage.
 This collector, helps the scintillator to acquire
secondary electrons.
 By changing the voltage the amount of secondary
electrons to be collected can be adjusted.
 This collector was originally developed by Everhart
and Thornley, so this detector can also be called as
E-T detector.

16.  When SEM is equipped with a strongly excited
objective lens for higher resolution a secondary
electron detector is placed above the objective lens.
 This type of detector is called TTL ie. (Through The
Lens ) detector.

17.  Image display unit- The out put signals from the
secondary electron detector are amplified and then
transferred to the display unit.
 Since the scanning on the display unit is
synchronized with the electron-probe scan,
brightness variation, which depends on the number
of the secondary electrons, appears on the monitor
screen on the display unit, thus forming a SEM
image.

18.  In general, the scan speed of the electron probe can
be changed in several steps.
 An extremely fast speed is used for observation and a
slow scan speed is used for saving of image.
 The image is recorded in a digital format (electrical
file), It is easier to process image and convenient to
send or receive image information.

19.  Vacuum system- The electron optical system and
the specimen chamber must be kept at a high
vacuum of 10-3 to 10-4 Pa.
 Thus , these component is evacuated by diffusion
pump. If a user desire an oil-free environment, a
turbo molecular pump may be used.

20. 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.

21.  Cleaning the surface of the specimen.
The proper cleaning of the surface of the specimen is
important, because the surface contains many
unwanted deposits, such as dust, mud, soil etc.
depending upon the source of the sample/specimen.

22.  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.
Fixation cab be achieved by :-
1. Perfusion or microinjection.
2. Immersions.
3. With vapours.

23.  Fixation is usually 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.

24.  Rinsing the specimen.
After the fixation step, the sample must be rinsed in
order to remove excessive fixatives.

25.  Dehydrating the specimen.
 All water must be removed from the samples because
the water would vaporize in the vacuum.
 The fixed tissue is then dehydrated. Because 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 is performed with a graded series of
ethanol or acetone.

26.  Drying the specimen.
 For SEM, a specimen is normally required to be
completely dry, since the specimen chamber is at
high vacuum.
 Otherwise the sample will be destroyed in the
electron microscope chamber.

27.  Mounting the specimen.
 After the specimen has been cleaned, fixed, rinsed,
dehydrated and dried, using an appropriate protocol,
specimen has to be mounted on the holder that can
be inserted into the scanning electron microscope.
 All samples must also be of an appropriate size to fit
in the specimen chamber and are generally mounted
rigidly on a specimen holder called a specimen stub.
 The dry specimen is usually mounted on a specimen
stub using an adhesive such as epoxy resin or
electrically conductive double-sided adhesive tape.

29. Charge-up

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

32.  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.

33.  Coating the specimen.
 The idea of coating the specimen is to increase the
conductivity of the specimen and to prevent the high
voltage charge on the specimen by conducting charge
to the ground.
 These specimen are coated with thin layer ie.20nm-
30nm of conductive metal.
 All metals are conductive and require no preparation
before being used.

34.  All non-metals need to be made conductive by
covering the sample with a thin layer of conductive
material.
 This is done by using a device called a "sputter
coater.”
 Conductive materials in current used for specimen
coating includes gold, gold-palladium alloy ,
platinum, osmium , iridium, tungsten, chromium
and graphite.

35. “Sputter Coater”

36. Advantages Disadvantages
1. Thermal conductivity is
increased.
2. Damaged to the sample
is reduced.
3. The quantity of
secondary electrons is
increased.
1. The shape & size of
nano particle is lost or
altered.
2. Specimen information
about elemental
composition & surface
potential may be lost.
Metal Coating

37. Why Images Are Visible?
 The SEM image appears as if it is been observed by
the naked eye.
 Interaction of electrons with specimens: when
e- enters the specimen, they are scattered with in the
specimen and gradually lose their energy.
 The scattering range of the electrons inside the
specimen is different depending on the electron
energy, atomic number, and the density of the
constituent atom.

38.  If the atomic number and density are larger, the
scattering range is smaller.
 If the electron energy is higher then the scattering
range is larger.

39. How Does SEM Works
 To further understand how does SEM works, we must
begin with the electrons. In a light microscope, light from
a source (usually an incandescent light) is focused
through lenses onto the sample. The image is
formed when the sample reflects and absorbs different
wavelengths of this light which is detected by our eyes
and formed into an image by our brains. An electron
microscope works in a similar fashion. Electrons from a
source are focused on the sample. These electrons reflect
off the sample, they are then picked up by an
electron detector and then processed into an
image which is projected onto a CRT that our eyes can
see.

40.  To begin our understanding of how an SEM works, let's
begin with the source of electrons, the electron gun.
 Most SEMs have what is called a hot cathode source,
usually a tungsten filament similar to that in a light bulb.
 When such a filament is heated by passing current
through it, it not only emits light, but an electron cloud
forms around the filament.
 Left on their own, they remain in the cloud and are
reabsorbed into the filament when the current is
removed.

41.  Placing a positively charged plate
(an anode) near the filament and
the electrons (being negatively
charged) will be attracted to it.
 Problem is, the electrons would
not be well directed and would
probably jump over to the anode
plate in a series of arcs.
 But by placing a negatively charged
cathode plate near the filament
(which they are repelled by) with a
hole in it and a positively charged
anode (which they are attracted to)
under this with another hole in it
and we have the makings of an
electron gun.

42.  The electron cloud is attracted to the anode plate
enough that they will travel through the hole in the
cathode. But in doing so, they gain enough speed
that most of them travel right through the hole in the
anode plate.
 Now we have an electron gun. The speed of the
electrons emitted from this gun is controlled by
the amount of potential (voltage) applied to the
cathode and anode plates.

43.  The electrons from the gun
come out in almost a
spray pattern, so we
may have a flow of
electrons, but this could
hardly be called a beam.
 We need lenses to control
the flow of electrons,
however, the glass lenses of
a light microscope will not
work. Instead, an
electron microscope uses
electromagnetic lenses.
spray pattern

44. An electromagnetic lens
An electromagnetic lens is a relatively
simple device. By applying current to
wire coiled around an iron cylindrical
core, a magnetic field is created which
acts as a lens.
The advantage of an electromagnetic
lens in an electron microscope is that
by varying the current through the
wires, the lens can have a variable
focal length.

45.  We now can arrange the electron gun and lenses in a
column mounted on a sample chamber.
 The condenser lens controls the size of the beam, or
the amount of electrons traveling down the column.
 The objective lens focuses the beam into a spot on
the sample. This is necessary to have an image in
proper focus.

46.  So far, the column we have designed will just focus
the electron beam into a spot on the sample.
 This is fine for welding or if we wanted the beam to
pass through the sample as in a TEM, but for an
SEM to work, we need the beam to scan.

47.  By placing sets of plates
around the beam and
varying the potential
between them, the
electron beam can be
deflected. If these plates
are attached to a scan
generator, the beam can
be made to scan lines
across the sample

48.  But this scan generator
is not only controlling
the scan coils, but is
also controlling the
beam of a CRT, the
image formed on the
CRT will be synched to
the electron beam
scanning the sample.

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

50. But how is the image formed?
 To understand this, we need to know what happens
when the electron beam interacts with the atoms of
the sample.

51.  The incident beam electrons (from the electron gun)
do not simply reflect off the sample surface.
 As the beam travels through the sample it can do
three things:
1. It can pass through the sample without colliding
with any of the sample atoms (matter is mostly
space).
2. It can collide with electrons from the sample
atoms, creating secondary electrons.
3. It can collide with the nucleus of the sample atom,
creating a backscattered electron.

52. How secondary e- are formed
 The incident beam is composed of
highly energized electrons. If one of
these electrons collides with a
sample atom electron, it will knock
it out of its shell. This electron is
called a secondary electron and is
weak in energy. If these secondary
electrons are close enough to the
sample surface, they can be
collected to form a SEM image.
 The incident beam electron loses
little energy in this collusion. In
fact, a single electron from the
beam will produce a shower of
thousands of secondary electrons
until it doesn't have the energy to
knock these electrons from their
shells.

53. How backscattered e- are formed
 If the incident beam collides
with a nucleus of a sample
atom, it bounces back out of
the sample as a backscattered
electron.
 These electrons have high
energies and because a
sample with a higher density
will create more of them,
they are used to form
backscattered electron
images, which generally can
discern the difference in
sample densities.
 Are used to determine crystal
structures and orientations of
minerals

54.  An electron detector is
placed in the sample
chamber. By having a 10
keV positive potential on
its face, it attracts the
secondary electrons
emitted from the sample
surface.

55. Detection of Secondary Electrons
 Secondary electrons hit
against the scintillator
for conversion into the
optical signal, which are
reconverted into
electrons on the
photoelectric conversion
face .
 These electrons are
accelerated with the
electric field and hit
against the first dynode.

56. Detection of Secondary Electrons
 These electrons are then
led to next dynode to
produce a large number
of secondary electrons.
 Thus the number of
secondary electrons
increases sequentially
and finally then taken
out as a signal current.

57. So how is the contrast formed?
 In secondary imaging mode, as the incident beam
scans across the sample's surface topography,
secondary electrons are emitted from the sample.

58.  If the beam travels into a
depression or hole in the
sample, the amount of
secondary electrons that can
escape the sample surface is
reduced and the image
processing places a
corresponding dark spot on
the screen.
 Conversely, if the incident
beam scans across a
projection or hill on the
sample, more secondary
electrons can escape the
sample surface, and the image
processing places a bright
spot on the screen.

59.  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.

60. Pollen and Stamens Wool fibers

61.  In backscattered imaging
mode, as the incident beam
scans across the sample's
surface topography,
backscattered electrons are
emitted from the sample.
 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.

62. 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.

63. Secondary image showing
surface morphology
Backscattered image
showing compositional
inhomogeneity
Image of a cement

64. Light’ region is made up predominantly of
Fe. (i.e. the heaviest element)
‘Grey’ region is made up predominantly of
Ca.
‘Dark’ region is made up predominantly of Si
and Al. (i.e. the lightest elements)

65.  Fig. 1 Light micrograph showing
the hard palatine mucosa.
Stratified squamous keratinized
epithelium (E), lamina propria
(*) and connective tissue papillae
are shown (arrow).
Fig. 2 SEM image showing the
surface of the palatine mucosa,
squamous epithelium (E) and
lamina propria (*).
Fig. 3 image of hard palatine
mucosa. Shows transverse
palatine plicae (*) and epithelial
projections (arrows).
Fig. 4 Shows elongated
protrusions on the surface of the
palatine mucosa (*).
Fig. 5 image showing polygonal
desquamated epithelial cells.
Fig. 6 Surface of polygonal
epithelial cells.

66. SEM Applications
 SEMs have a variety of applications in a number of
scientific and industry-related fields, especially
where characterizations of solid materials is
beneficial.
 In addition to topographical, morphological and
compositional information, a Scanning Electron
Microscope can detect and analyze surface fractures,
provide information in microstructures, examine
surface contaminations, reveal spatial variations in
chemical compositions, provide qualitative chemical
analyses and identify crystalline structures.

67. Presence of extensive areas of
resorption can be noticed on the
lingual aspect of all roots.
Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay
J. Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009
A scanning electron microscopic study of the patterns of external root resorption
under different conditions

68. Mandibular permanent molar
undergoing root resorption due to
an associated periapical granuloma
Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay
J. Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009
A scanning electron microscopic study of the patterns of external root resorption under
different conditions

69. Mandibular first premolar undergoing
pressure resorption during the course of
orthodontic treatment
Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay
J. Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009
A scanning electron microscopic study of the patterns of external root resorption
under different conditions

70.  Human blood was obtained
by venous puncture.
 The RBCs were isolated by
centrifugation.
 Fixation- 1% glutaraldehyde
 Washed- phosphate buffer.
 Mounting
 Sputter coating- with gold

71. SCANNING
ELECTRON
MICROSCOPIC
IMAGE OF THE
TONGUE

72. Scanning electron micrographs of the early
human embryo

73.  SEMs can be as essential research tool in fields such
as life science, biology, gemology, medical and
forensic science, metallurgy.
 In addition, SEMs have practical industrial and
technological applications such as semiconductor
inspection, production line of miniscule products
and assembly of microchips for computers.

74. SEM Advantages
 Advantages of a Scanning Electron Microscope
include its wide-array of applications, the detailed
three-dimensional and topographical imaging and
the versatile information garnered from different
detectors.
 SEMs are also easy to operate with the proper
training and advances in computer technology and
associated software make operation user-friendly.
 This instrument works fast.

75. SEM Disadvantages
 The disadvantages of a Scanning Electron
Microscope start with the size and cost.
 SEMs are expensive, large and must be housed in
an area free of any possible electric, magnetic or
vibration interference.
 Maintenance involves keeping a steady voltage,
currents to electromagnetic coils and circulation of
cool water.

76.  Special training is required to operate an SEM as
well as prepare samples.
 The preparation of samples can result in artifacts.
The negative impact can be minimized with
knowledgeable experience researchers being able to
identify artifacts from actual data as well as
preparation skill. There is no absolute way to
eliminate or identify all potential artifacts.

77.  In addition, SEMs are limited to solid, inorganic
samples small enough to fit inside the vacuum
chamber that can handle moderate vacuum pressure.
 Finally, SEMs carry a small risk of radiation
exposure associated with the electrons that scatter
from beneath the sample surface.

78. Difference between SEM and TEM
TEM SEM
Based on transmitted electrons. scattered electrons
focuses on internal composition. sample’s surface
image 2D image 3D image
shows morphology,
crystallization, stress or
even magnetic domains.
morphology of samples.
sample cut thinner no such need
resolution much higher low
Size of sample only small amount of
sample can be analysed
allows for large amount of
sample to be analysed
pictures are shown on fluorescent screens is shown on monitor
Sample preparation Is very important need special sample
preparation.

79. References
1. Theory & practice of histological techniques: John
Bancroft, M Gamble.
2. Scanning Electron Microscopy, Dr H. Bagshaw
3. Introduction to SEM. By Rodney Herring
4. Hortolà, P. (2010). "Using digital colour to increase the
realistic appearance of SEM micrographs of
bloodstains". Micron 41 (7): 904–908.
5. "Introduction to Electron Microscopy" . FEI Company.
p. 15. Retrieved 12 December 2012
6. Ravindran Sreeja,Chaudhary Minal. -J. Appl. Oral
Sci. vol.17 no.5 Bauru Sept./Oct. 2009-A scanning
electron microscopic study of the patterns of external
root resorption under different conditions

80. THANK YOU