Electron microscopy

Introduction to microscopy
Development of microscope
Types of microscopes
Electron microscope

Definition
Development of electron microscope
Configuration
Basic working principle
Types of electron microscope
Processing of tissue for electron microscopy
Advantage and disadvantage of electron microscopy

Introduction to microscopy
The word microscope is derived from the Greek
word mikros ( small) and skopeo (look at). Literally a
microscope can be defined as an instrument used to
view objects that are not visible to our naked eye.
No body exactly knows who invented the
microscope. One of the earliest instrument as
microscope was made by the Dutchman Antony van
Leeuwenhoek(1632- 1723) and consisted a powerful
convex lens. With this Leeuwenhoek was able to
magnify objects up to 400X. With this he discovered
protozoa, spermatozoa and bacteria and was also
able to classify red blood cells by shape.

Development of microscope
From the dawn of science there has been an
interest in being able to look at smaller and
smaller details of world around us.
Biologist wanted to examine the structure
of cells bacteria viruses
Chemist wanted to see molecular details of
elements and geologist wanted to study the
details of rocks and minerals.
So all these elements led into the
development of microscope.

Types of microscopes
Most microscopes can be classified as one of three
basic types:
Optical microscope
Charged particle microscope( electron/ ion microscope)
Scanning probe microscope

Optical microscope uses visible light and transparent

lenses to see objects.
Electron and ion microscopes uses a beam of charged
particles instead of light and use electromagnetic lens
to focus the particles.
Scanning probe microscopes use a physical probe (a
very small, very sharp needle) which scan over the
sample in contact or near contact with the surface.

Electron microscope
Electron microscope are scientific
instruments that use a beam of
highly energetic electrons to
examine objects on a very fine scale.

Development of electron
microscope
One of the limiting factor in Leeuwenhoeks
microscope was the single convex lens which could
not provide sufficient magnification.
This problem was solved by addition of another
lens to magnify the image produce by the first
lens. This compound microscope- consisting an
objective lens and an eye piece together with a
means of focusing, a mirror or source of light and a
specimen table for holding and positioning the
specimen- is the basis of light microscope and
forms the basis of present compound microscopes.

Development of electron microscope

The modern light microscope has a magnification of about 1000X
and enables the eye to resolve objects by 200nm. Objects
separated by less than 200nm could not be separated by the LM.
This became a major drawback of LM.
Soon scientists found out that the resolving power of the
microscope was not only limited by the number and quality of the
lenses but also by the wavelength of the light use for illumination.
LM uses light of visible range( wavelength 400- 700nm) to
illuminate the specimen. This radiation limited the resolving power
of the microscope.
Using the light of shorter wavelength( ultraviolet) gave a little
improvement but not enough.
Immersing the specimen and the front of the objective lens in
medium with high refractive index( such as oil) gave another small
improvement but the resolving power was not improved more
than 100nm.

Development of electron
microscope
In 1920 it was discovered that accelerated
electrons behave in vacuum much like light.
They travel in straight lines and have wave
like properties with a wavelength that is about
1,00,000 times shorter than that of visible
light.
Furthermore, it was found that electric and
magnetic fields could be used to shape the
path followed by the electrons similar to the
way glass lenses are used to bend and focus
the visible light.

Wavelength Vs resolution

Development of electron
microscope
Ernst Ruska at the university of Berlin combined these
characteristics and built the first transmission electron
microscope in 1931. For this and subsequent work in the
subject he was awarded the Nobel prize for physics in
1986.
His first electron microscope used two electromagnetic
lenses and three years later he added a third lens and
he demonstrated a resolution of 100nm twice as good
as that of light microscope.
Today electron microscope have reached a resolution s
of better than 0.05nm, more than 4000 times better
than a typical light microscope and 4000000 times
better than the unaided eye.

Basic configuration of electron

microscope
There are four main components in
an electron microscope.
An electron column
Vacuum system
Necessary electronics
Electromagnetic lenses
High voltage generator source

Control software

Basic configuration

The electron gun

Three main types of electron sources are
used in electron microscope:
1. Tungsten
2. Lanthanum hexaboride
3. Field emission gun.

Among these source the tungsten is the

most economical and is also satisfactory.
The tungsten gun comprises a filament a
Wehnelt cylinder and an anode. These three
together form a triode gun, which is a very
stable source of electrons.
The tungsten filament is a hairpin in shaped
and heated up to 2700o C.

The electron gun

The anode has a hole in it so that an electron beam
in which high speed electrons emerges and directed
down the column.
The Wehnelt cylinder serves to shape the electron
beam.
A large potential is applied between the tungsten
filament and the anode. This gives raise to electron
beam with very high speed.
The wavelength of electron is inversely proportional
to the its velocity. So a high speed electron has very
small wavelength which provides a better resolution
power.

The electron gun

The electromagnetic lenses

These lenses are based on the fact that the
electrons can be deviated and can be focused
at a point by applying the magnetic field.
The power of the electromagnet can be varied
by passing the amount of current passing
through it.
High current-large magnetic field-high power
low current-small magnetic field low power

Ocular lens Vs
electromagnetic lens
Optical lens- fixed focal length
Electromagnetic lens- variable focal length
Optical lens- variable position
Electromagnetic lens- fixed position
Optical lens- fixed power-depends upon
focal length
Electromagnetic - variable power-depends
upon the
current passing through

magnitude of

Electromagnetic lens
When an electric
current is passed
through the coils (C),
an electromagnetic
field is created
between the pole
pieces (P). By varying
the current through the
coils, the strength of
the field, and thereby
the power of the lens,
can be varied.

Vacuum
Electrons behave like light only when they
are in vacuum. So unless and until the
whole system is under vacuum, the whole
apparatus is of no significance.
Various vacuum levels are produced inside
the column. The highest vacuum is around
the specimen and in the source; a lower
vacuum is found in the projection and
fluorescent screen chamber.
Vacuum can be as high as 10 -8Pa.

Principle

The electron beam produced

from the electron gun (heated
tungsten filament) is
condensed into a nearly
parallel beam at the specimen
by the condenser lenses. This
electron beam passes through
the specimen and is collected
and focused by the objective
lens. The image formed by
the objective is taken up by
the projector lens and is
finally projected as a real and
magnified image on to the
fluorescent screen.
The entire electron path from
the electron gun to the
fluorescent screen should be
under vacuum (otherwise the

What happens in the specimen during

the electron bombardment?
Some of the electrons are ABSORBED as a function of the

thickness and composition of the specimen; these cause what is

called AMPLITUDE CONTRAST in the image.
Other electrons are SCATTERED over small angles, depending on
the composition and structure of the specimen; these cause what
is called PHASE CONTRAST in the image.
In crystalline specimens, the electrons are scattered in very
distinct directions that are a function of the crystal structure; these
cause what is called DIFFRACTION CONTRAST in the image.
Some of the electrons are deflected through large angle or
reflected (backscattered) by sample nuclei.

What happens in the specimen during

the electron bombardment?
The impinging electrons can knock electrons from sample atoms
which escape as low energy secondary electrons.
The impinging electrons may cause specimen atoms to emit Xrays whose energy and wavelength are related to the specimens
elemental composition; these are called characteristic X-rays. The
impinging electrons cause the specimen to emit photons(or light);
this is called cathodoluminescence.
Finally, transmitted beam electrons can be counted and sorted by
an energy loss spectrometer according to the amount of energy
they have lost in interactions with the specimen. The energy loss
carries information about the elemental, chemical, and electronic
states of the sample atoms

What happens in the specimen during

the electron bombardment?
In a standard TEM, amplitude contrast
is the primary contrast mechanism
for non-crystalline (biological)
specimens, while phase contrast and
diffraction contrast are the most
important factors in image formation
for crystalline specimens (most nonbiological materials).

Tissue preparation
In routine histopathology the section thickness is around
3-4 microns thick.
A electron beam is capable of penetration tissue of
around 100nm. So to obtain a high quality image and to
optimize the resolution of the instrument it is necessary
to cut the sections to a thickness of around 80nm.
To prepare the section thickness at this level the tissue
needs to be embedded in extremely rigid materail. And
routine paraffin embedding cannot provide this rigidity.
So the embedding procedure is carried out in special
embedding resin.

Fixation
Should begin as soon as the specimen is
taken out of the body. Delay in fixation brings
about the postmortem changes in the tissue.
Volume of fixative: 10 times the volume of
tissue.
Once in fixative the tissue is grossed and cut
into the size of 1mm3. This tissue is then
suspended in the fixative container to ensure
that the tissue is completely submerged in
the fixative.

Fixation
The standard protocol for fixation involves primary
fixation with glutaraldehyde followed by secondary
fixation in osmium tetroxide.
Glutaraldehyde:

effective from 1 4%.

2.5% the simplest to prepare from 25% stock solution commercially
available.

Osmium tetroxide is commonly used at a concentration of 1 2%.

Temperature:- Fixation at room temperature enhances the

penetration rate and decreases the duration of fixation but also
increases the risk of autolytic changes. So many practitioners
prefer fixation at cold temperature. Osmium tetroxide is used at
room temperature.

Duration: 2 - 6 hours primary fixation in glutaraldehyde

60- 90 minutes secondary fixation in
osmium
tetroxide

Fixation
Glutaraldehyde
Glutaraldehyde brings cross linking of
amino group of lysine and amino acids
through the formation of pyridine
intermediates and thus helps in
preservation of proteins.
At times formaldehyde paraformaldehyde
and other aldehyde combination (such as
paraformaldehyde, 2%
glutaraldehyde2.5%) are also used.

Fixative
2.5% buffered glutaraldehyde
Composition:
25% glutaraldehyde stock solution: 10ml
0.1M phosphate buffer: 90 ml

Method of preparation
Combine glutaraldehyde and phosphate
buffer in the proportion indicated and
mix properly

Fixation
Osmium tetroxide
Used to preserve the lipids.
Good primary fixative but since has slow penetration
as primary fixative it may induce autolytic changes in
the tissue. And further the penetration rate of
osmium tetroxide in pre fixed tissue is better.
Care should be taken while preparing the osmium
tetroxide solution from the commercial osmium
tetroxide crystals. PPE should always be worn.
Osmium tetroxide can be prepared as aqueous
solution although it can be made in same buffer used
to prepare the primary fixative.

Fixative
2% aqueous osmium tetroxide
Composition
Osmium tetroxide (crystal) :1gm
Distilled water: 50ml

Method of preparation
Combine osmium tetroxide and water in the proportion as
indicated and mix properly.
Then store in double walled glass container to prevent the leakage
of fumes of osmium tetroxide and can be stored up to 1 year .
As a working solution 1% OsO4 is prepared by mixing the solution
with equal volume of distilled water.

The reagent is readily reduced by dust and light. So

glassware that has been acid cleaned and thoroughly rinsed
with distilled water should be used.

Fixative
Specimens fixed in glutaraldehyde should be
washed thoroughly in buffer before transferring
it into secondary osmium tetroxide solution.
Inadequate washing will cause the interaction
between the two fixative and may lead to
precipitation of reduced osmium.
After fixation the tissue is washed in buffer to
remove excess of fixative.
Additionally the tissue after fixation can be
immersed in 2% uranyl acetate. This provides
contrast and also improves preservation.

Dehydration
Most common embedding agent in electron
microscopy is the epoxy resins which is
completely immiscible with water. Thus
dehydration should be complete for electron
microscopy.
Most commonly used dehydrants: Acetone
Ethanol
Dehydration is carried out by passing the
specimen through graded dehydrants. Sudden
extreme change in dehydrant concentration
can produce damage in the specimen.

Dehydration
Acetone- can precipitate the uranium salts if
used for the specimen which has been
immersed in 2%uranium solution after fixation.
Ethanol-requires propylene oxide as a transition
solvent to facilitate resin infiltration in
embedding.
Commercial absolute ethanol normally contains
a small percentage of water which may restrict
infiltration of the resins. So in order to produce
complete dehydration special anhydrous
ethanol should be used.

Infiltration and Embedding

Infiltration and embedding follows
dehydration
There is gradual infiltration of the resin into
the specimen.
If transitional solvent is used the schedule is:
50:50(T:R)- 1 hour
25:75(T:R)-1 hour
100%(R)- 24 hour

Gentle agitation improves the rate and

quality of infiltration.

Infiltration and Embedding

Once infiltrated the tissue are placed
in embedding mould containing the
resin.
Embedding mould made up of
polyethylene are recommended as
polyethylene is unreactive with the
resins. And further the embedded
tissue can be easily removed from
the mould by bending the mould.

Infiltration and Embedding

Epoxy resins
Medium of choice.
Mode of action: these resins contains a characteristic
chemical group in which an oxygen and two carbon atoms
bond to form three membered ring called the EPOXIDE.
Cross linking between these groups creates a three
dimensional polymer of great mechanical strength.
Once polymerized it is permanent and can also withstand
the electron beam and vacuum in electron microscopy.
These resins produce very less shrinkage and have a
property of even polymerization which helps in uniform
and easy sectioning.

Infiltration and Embedding

Epoxy resins
Epoxy resins usually consists four ingredients:
Monomeric resin
Hardener
An accelerator
A plasticizer

The hardness and flexibility of blocks and polymerization

time can be manipulated by varying the amount of the
individual component.
Examples of epoxy resins:
Ardalite (Glauert and Glauert, 1958)
Expon ( Luft1961)
Spurrs resin (spurr 1969)

Infiltration and Embedding

Acrylic resins
Original synthetic media developed for EM.
Derived from methacrylic acid and acrylic acid.
Rapidly infiltrates the tissue but has unreliable
polymerization and these resins are relatively
unstable in electron beam.
Acrylic resins react by free radical polymerization
which can be initiated using light heat or chemical
accelerators.
Examples of acrylic resins:
LR white
LR Gold

Ultramicrotomy
Knives: diamond and glass knives of best quality are
used.
After embedding the blocks are trimmed and made
ready for microtomy. This trimming can be done
manually using a razor or by a microtome.
Then semi thin sections(0.5- 1um) are produced.
These sections are meant to screen for specific
features and to select an area for thin sectioning.
These sections are floated in plastic or metal trough
attached directly to the knife. Trough fluid usually
comprises of distilled water. Alternatively 10-15%
acetone or ethanol can also be used.

Ultramicrotomy
The semi thin sections are then transfereed
on a glass slide and dried on a hot plate at
70-800C.
These sections can be examined using
phase contrast microscopy or can be stained
and viewed by bright field microscopy.
Stains include:
Methylene blue
Crystal voilet
Azure B

Ultramicrotomy
Then ultrathin sections are cut off
and floated directly on to the
floation bath.
These sections are then collected
on special specimen grids.
Grids:
3.05mm diameter
Made up of nickel or gold silver,
palladium
A large pattern and mesh sizes are
availabel. Common squaremesh.
The choice of grid depends upon
two factor .
Support for the section(better with
small mesh)
Relative propotion of exposed
section(better with large mesh)

 To collect the section the grids are

immersed in the flotation bath and
sections are collected on to them.
These grids are then placed over a
filter patere in a lidded container
such as a petri dish and allowed to
dry completely.

Staining

Application
Life sciences
Electron microscopy is being used today in research laboratories around the world to
explore the molecular mechanisms of disease, to visualize the 3D architecture of
tissues and cells, to unambiguously determine the conformation of flexible protein
structures and complexes, and to observe individual viruses and macromolecular
complexes in their natural biological context.
Structural biology 3D techniques, electron tomography and single particle
analysis, allow researchers to derive important information regarding protein
domain arrangements and, in some cases, to trace individual polypeptide chains.
The combination of electron microscopy construction with X-ray crystallography
and NMR spectroscopy enables even greater structural detail by fitting atomic
scale structural models into the EM density map.
Cellular biology High-resolution cryo microscopy avoids the alterations caused by
conventional preparation techniques to allow imaging of cell membrane structures
and sub-cellular morphology in fully hydrated conditions.
Tissue biology An electron microscopes ability to provide high resolution ultra
structural imaging over large areas and volumes of tissues or cells is invaluable in
discerning critical relationships among components of biological systems across
large differences in spatial scale.
Biomaterials The properties of biomaterials and nano particles are highly
dependent on structural characteristics that are readily observed using electron
microscopy.

Application
Research
Electron microscopes are being applied successfully in the pursuit of a deeper
understanding of the structure-property-function relationships in a wide range of
materials and processes such as next generation fuel cell and solar cell technologies,
catalyst activity and chemical selectivity, energy-efficient solid-state lighting; and
lighter, stronge.r, and safer materials.
3D nanocharacterization Nanocharacterization moves to a new level with STEM,
TEM, and DualBeam tomography affording 3D visualization at the nanoscale.
Analytical techniques such as electron backscatter diffraction (EBSD), X-ray
microanalysis (EDSX), and energy-filtered TEM (EFTEM) can also be extended to three
dimensions, giving a world of new information relating structures to properties.
In situ nanoprocesses The electron microscope becomes a lab ina chamber with
ESEM and ETEM technologies allowing dynamic control over temperature, pressure,
and gas type for in situ nanoprocess investigations. Researchers can visualize and
correlate the structure, property, and function of materials undergoing chemical and
physica lprocesses such as catalysis, oxidation, reduction, polymerization,
deformation and thermally induced phase transformations.
3D nanoprototyping Nanoprotyping is a fast, simple way to design, fabricate, and
test small-scale structures and devices usingan electron beam or a focused ion
beam. Site-specific milling,lithography, or chemical vapor deposition can all be
carried out at the nanoscale to deliver high-quality 3D nanoprototyped structures

Application
Industry
Natural resources Mining companies use automated electron
microscopyto analyze millions of micro-scale features in an automated,
objective, quantitative, and rapid manner. The results compliment bulk
chemical assays and together they are used to maximize metal recovery
and guide decisions in exploration, mining, mineral processing, and metal
refining. In oil and gas exploration similar analyses provide quantitative
lithotype and porosity characteristics of reservoir, seal, and source rocks.
The results enhance and validate seismic, wireline,and mud logs, providing
input into geological models and reducing risk in exploration and extraction.
Forensics Forensic science uses electron microscopy to analyze criminal
evidence such as gunshot residue, clothing fibers, handwriting samples, and
soil.
Other automated particle analysis Inorganic particles both natural
and manmade including soil, coal, cement, fly ash, and airborne dust, can
be analyzed to provide a more detailed understanding of the impact of
waste and pollution on the environment and health.

Application
Electronics
In laboratories and production facilities for semiconductor, solar,
micro-electro mechanial system (MEMS) labs, and data storage
devices, electron and ion microscopy provide the high resolution
imaging and analysis required to develop and control
manufacturing processes.
Circuit edit Engineers use a FIBs precise milling and material
deposition to rewire integrated circuits to check design
modifications without having to repeat the lengthy and expensive
manufacturing process.
Failure analysis The Dual Beams ability to cross-section
subsurface defects and quickly prepare site-specific thin section
sample for high resolution imaging in S/TEM allows engineers to
determine the root causes of manufacturing defects.
Metrology and process control As the dimensions of
microelectronic devices have shrunk beyond the resolving power
of optical microscopes (and in some cases, beyond that of SEM as
well), S/TEM provides critical feedback needed to control
manufacturing processes.