ELECTRON MICROSCOPY

Presented by
ANISH KUMAR
CHUDHARY
METT/61/15
 CONTENTS
• Introduction
• Working principle
• Type of electron microscope
• Sample preparation
• Conclusion
 Microscopy
Introduction:
Microscopy is the technical field of using
microscopes to view objects and areas of objects
that cannot be seen with necked eye.
There are three branches of microscopy :-
Optical microscopy
Electron microscopy &
Scanning probe microscopy
 Optical microscopy
• Optical microscopy involves visible light transmitted
through or reflected from the sample through a single
lens or multiple lenses to allow a magnified view the
sample.
• The resulting image can be detected directly by the
eye ,imaged on a photographic plate.
Limitations
• This technique can only form dark image .
• Diffraction limits resolution to approximately 0.2
micrometers
 Electron Microscopy
• Electron microscopy is a technique for obtaining high
resolution images of biological and non biological specimen.
• It is used in biological research to investigate the detail
structure of tissues, cells, organelles and macromolecular
complexes.
• The high resolution of EM images results from the use of
electrons(which have very short wavelengths) as the source of
illuminating radiation.
• Advantage of electron microscopy
 Most important advantage of electron microscopy is the
ability to produce power magnification.
 It offers a higher resolution than optical microscopy.
 Working principal of electron
microscopy
The basic steps involved in all Ems are the following:
• A stream of electrons is formed in high vacuum (by
electron guns).
• This stream is accelerated towards the specimen (with a
positive electrical potential) while is confined and
focused using metal apertures and magnetic lenses into
a thin, focused, monochromatic beam.
• The sample is irradiated by the beam and interactions
occur inside the irradiated sample, affecting the
electron beam.
• These interactions and effects are detected and
transformed into an image.
Design of EM
 Major Components of EM
 Electron Gun
 Vacuum system
 Lenses
 Aperture
 Electron Gun
• The electron gun generates the electron beam.
• It is usually positioned in the top of the instrument column.
• In the image sequence below the gun assembly is lifted off
and moved aside to show how the electron emitter is
replaced.
• The emitter is seated within a cone-shaped Wehnelt cylinder
and the beam travels out of the small central hole.
 Electron Gun continue
• Electron guns can be classified into two types
• Thermionic gun
• Field emission gun
Thermionic Gun:
The typical thermionic electron gun consists of three parts,
the filament, Wehnelt cap and the anode.
 electrons are emitted from a heated wire (filament).
 due to the negatively charged Wehnelt cylinder electrons are only emitted
from the tip of the wire
 therefore the electron beam is easier to focus.
 The filament can be made of tungsten or laB6.
 If filaments made of LaB6 generate stronger currents
 (more electrons).
field emission:
 if the negative voltage applied to the cathode is large enough, electrons are
emitted from it even without heating as a result of quantum mechanical
tunneling.
 Field emission cathodes are made of tungsten.
 Lenses
• TEM lenses are operate electrostatically or magnetically.
• Within the column the electromagnetic lenses shape the
electron beam, which travels in a spiral trajectory.
• Each lens is constructed of a coil of copper wire through
which a current runs and creating a magnetic field.
• There is a hole in the center through which the beam travels.
• The inside of the column is maintained under vacuum so that
the density of molecules which can interfere with the electron
beam are minimized.
• To achieve this, a system of vacuum pumps are attached to
the TEM column.
 Need of Vacuum System
In an electron microscope, especially in a TEM,
vacuum must be generated in order to prevent
1. scattering of electrons
2. Discharges between high-voltage components.
3. Oxidation of filament.
• It is also used to increase the average distance of
moving particles.
 Aperture
• Aperture are metallic plates
• It is consist of a small metallic disc.
• it is use to prevent electron passing through the disc.

• The objective aperture is the first aperture situated

below the sample plane.
• It is used to increase contrast and is introduced into the
column for bright field and dark field imaging.
 Type of electron Microscopy
• There are two type of electron
microscopy
Transmission electron microscope (TEM)
Scanning electron Microscope (SEM)
 Scanning Electron Microscope (SEM)
• A scanning electron microscope is a type of electron
microscope that produces images of a sample by scanning
the surface with a focused beam of electrons.
• The electron interact with atoms in the sample, producing
various signals that contain information about the surface
topography and composition of the sample.
• The electron beam is scan in raster pattern and position of
the beam is combined with the detected signals to
produce an image.
• Use backscattered electron or secondary electron beam.
• SEM can achieve resolution better than 1nanometer.
Ray diagram of SEM
 Sample Preparation For SEM

• Cleaning the surface : Remove all the dust , silt , media components
• Stabilizing the specimen : Stabilization is typically done with fixatives.
• fixation by immersing the specimen in a 1.5% glutaraldehyde solution prepared in
0.1 M cacodylic acid buffer (pH 7.3)
• Rinsing the specimen : After the fixation step, samples must be rinsed in order to
remove the excess fixative.
• Dehydrating the specimen : removal of water from the sample.
It is typically performed with either a graded series of acetone or ethanol.
• Drying the specimen :The scanning electron microscope operates with a vacuum.
Thus, the specimens must be dry or the sample will be destroyed in the electron
microscope chamber.
• Mounting the specimen : After the cleaned, fixed, rinsed, dehydrated, and dried
specimens must be mounted on a holder that can be inserted into the scanning
electron microscope.
• Coating the specimen : coating the specimen is to increase its conductivity in the
scanning electron microscope and to prevent the build-up of high voltage charges
on the specimen by conducting the charge to ground.
 Transmission Electron
Microscope(TEM)
• In this microscope, use a beam of electron which is
transmitted through a specimen to form an image.
• Specimen is most often an ultra thin section less than
100 nm thick.
• An image is formed from the interaction of the
electrons with the sample as the beam is transmitted
through the specimen.
• It is capable to form higher resolution microscope
than light microscope.
Ray Diagram For TEM
 Sample preparation for TEM
• For biological samples(Bone, shell etc.)
• The main aim of biological sample preparation is the
 generation of ultra-thin samples transparent for
electron beams
 Contrast enhancement with stains containing heavy
metals
Steps of biological sample preparation:
1. Cleaning: Remove the dust from the specimen.
2. Fixation: with glutaraldehyde in most cases
3. Dehydration:
 water is removed by placing the sample into a series of increasing
ethanol or acetone concentrations.
 Dehydration is required because water would quickly evaporate in
the vacuum of TEM damaging the sample.
 In addition, epoxy resin is hydrophobic, and it can penetrate the
sample if water is removed.
4. Embedding: Samples are impregnated by epoxy resin so as to
make them solid for cutting.
5. Cutting with an ultramicrotome: a glass or a diamond blade cuts slices
of ~ 100 nm thickness,
which are allowed to swim on the surface of water.
6. Samples floating on water are placed on a grid made of copper.
1. The grid is covered by a material transparent for electron beams providing support
for the sample.
2. The samples are placed into the electron microscope on the grid. Only those parts
of the sample can be investigated with the electron microscope that are above the
holes of the grid.

7. Staining: it can be performed after putting the sample on the grid or just after
fixation.
 Staining is required because biological samples provide very weak contrast
(homogenous thickness, homogenous distribution of atomic index).
 During staining the sample is treated with material containing atoms with high
atomic number.
 Imaging
There are three types of images generate in TEM:
1) Bright field image
2) Dark field image
3) Diffraction image
Bright field image
Bright field image appears as a shadow of the specimen.
In the bright field image the objective aperture is used to select
the unscattered electron beam.
This aperture enhances the contrast in the image.

Zinc oxide crystals: bright field image

.
 Imaging continue
Dark field image
 Dark field images are produced by scattered electrons (i.e. only
selected electrons are used to form the image).
 Regions where no scattering occurs, such as where the primary
electron beam passes straight through the sample, appear
black(e.g. in areas around the sample).
 This kind of imaging is useful in studying crystal defects, and for the
imaging of specific crystallographic phases.

fig: Dark field image of zinc oxide

 Diffraction pattern
 TEM provide information about its sample structure. This is
because a crystal lattice acts as a diffraction grating.
 Interference patterns are produced when the electron beam as
it travels out from the lattice and
 These can be projected as an image of regular dots or rings.
 Imaging continue
Diffraction images:
 Diffraction images are the result of Bragg scattering as the
beam passes through a crystalline sample.
 If a “selected area diffraction aperture” is inserted to delimit
the region of interest, then an image created below the
sample (in the region called the back focal plane) is seen as an
array of dots (or a set of diffuse rings).
 This informs the viewer about the crystal structure of the
sample.

Nd13cao7
 Diffraction pattern continue
• Direction and distance from the center of the screen at
which diffraction spot appears is determined by Bragg’s law
2dsin(θ)=nλ
Where,
λ= wavelength of electron
d= spacing of the crystal plane
It is the function of miller indices (h,k,l)
as well as the lattice parameter(S)
d= a/( (𝒉² + 𝒌² + 𝒍²)
θ= diffraction angle , which gives the information about
crystal orientation.
n= number of integer
 Difference between TEM and SEM
TEM
 Electron beam pass through
the sample.
 Based on transmitted
electron .
 Produce two dimensional
black and white image.
 Very thin sample is
required.
 Skilled person required for
operate
 High resolution
SEM
 Electro beam scans over
the surface of the sample.
 Based on scattered
electron.
 Produce three
dimensional black and
white image.
 Thickness of the sample is
variable
 Low resolution than TEM
 Conclusion
 From the all discussion we can conclude that electron
microscopy technique is the very useful to view the high
resolution and magnifying image of the ultra thin objects
such as biological and non biological.
 Electron microscopy technique is able to form the two
dimensional and three dimensional image and imagese
are black and white.
 By this technique we easily find out the defect present in
the materials.
 In this technique beam of electron is source of image of
the objects.
 Sample preparation of these technique is very long
therefore it is not suitable for daily laboratory practice.
 References
• http://en.wikipedia.org/wiki/cryofixation
• http://www.ammrf.org.au/myscope/tem/introdu
ction/
• https://cfamm.ucr.edu/documents/fei-em.pdf
• http://www.formatex.org/microscopy3/pdf/pp12
2-131.pdf
• http://mbft.hu/assets/sejtanalitika/electronmicro
scope.pdf
• https://nios.ac.in/media/documents/dmlt/HC/Le
sson-19.pdf
Thank you