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The electron microscope uses electrostatic electro
magnetic lenses to control the electron beam and focus it to
form an image.
These electron optical lenses are analogous to the glass lenses
of a light optical microscope.
Electron microscopes are used to investigate
the ultrastructure of a wide range of biological and inorganic
specimens including microorganisms, cells,
large molecules, biopsy samples, metals, and crystals.
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Types
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
Reflection electron microscope (REM) Scanning transmission electron microscope (STEM)
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Principles of Electron Microscope Electron microscopes use "electron beams" which have
wavelengths much shorter than that of light. These apparatus emit an electron beam toward the object to
be investigated, detect the electrons which pass through, are
reflected from or emitted from the object, and create a
picture.
The brighter and finer the electron beam, the higher the level
of observation of the objectsinternal details including atomic
arrangement.
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The wavelength of electrons is less than 1/100,000 of that of
visible light, or 1 picometer (pm), which is 0.001 nm.
Therefore, theoretically, the resolution of electronmicroscopes can be less than several picometers. However,
the resolution obtainable for an electron microscope is
restricted to approximately 100 pm by lens aberrations.
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The electron lens uses a magnetic field generated by an
electrical current in a coil to converge the electrons
The electrons being converged in the magnetic field generated by
the coil
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Electrical current is passed through the coil.
A magnetic field is generated
Put electrons in the magnetic field; then they travel parallel
to the optical axis
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The direction of the magnetic field is downward. The electrons are moving
toward the back; this means that the electrical current is towards the front
Let B the magnetic field and I the electrical current, then electrons are
subject to the force F according to the Flemingsleft-hand rule
That electrons are circling around the optical axis
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The direction of this force is toward the optical axis.
As a result of this process, all electrons converge at a single point.
When the electrons rotate, they are now subjected
to the magnetic field parallel to the optical axis.
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The electron microscope lens system is made up of several electron
lenses.
Electron microscopes can observe much smaller objects that cannot be seen
by optical microscopes.
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Sample preparation Materials to be viewed under an electron microscope may
require processing to produce a suitable sample. The technique required varies depending on the specimen
and the analysis required:
Chemical fixation chemical cross linking of proteins
with aldehydes such as formaldehyde and glutaraldehydeand lipids with osmium tetroxide.
Negative stainmixed with a dilute solution of an electron-
opaque solution such as ammonium molybdate, uranyl
acetate (or formate), or phosphotungstic acid.
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This mixture is applied to a suitably coated EM grid, blotted,
then allowed to dry.
Viewing of this preparation in the TEM should be carried outwithout delay for best results.
Negative staining is also used for observation of
nanoparticles.
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Cryofixation freezing a specimen so rapidly, to liquid
nitrogen or even liquid helium temperatures, that the water
forms vitreous (non-crystalline) ice. This preserves the specimen in a snapshot of its solution
state.
With the development of cryo-electron microscopy of
vitreous sections, it is now possible to observe samples fromvirtually any biological specimen close to its native state.
Dehydration freeze drying, or replacement of water with
organic solvents such as ethanol or acetone, followed
by critical point drying or infiltration with embedding resins.
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Embedding after dehydration, tissue for observation in the
transmission electron microscope is embedded so it can be
sectioned ready for viewing.
To do this the tissue is passed through a 'transition solvent'
such as Propylene oxide and then infiltrated with
an epoxy resin such as Araldite.
Embedding, materials after embedding in resin, the
specimen is usually ground and polished to a mirror-like finish
using ultra-fine abrasives.
The polishing process must be performed carefully to
minimize scratches and other polishing artifacts that reduce
image quality.
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Sectioning produces thin slices of specimen,
semitransparent to electrons.
These can be cut on an ultramicrotome with a diamond knifeto produce ultra-thin slices about 6090 nm thick.
Staining uses heavy metals such
as lead, uranium or tungsten to scatter imaging electrons and
thus give contrast between different structures. Typically thin sections are stained for several minutes with an
aqueous or alcoholic solution of uranyl acetate followed by
aqueous lead citrate.
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Freeze-fracture or freeze-etch a preparation method
particularly useful for examining lipid membranes and their
incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly
(cryofixation), then fractured by simply breaking or by using a
microtome while maintained at liquid nitrogen temperature.
The cold fractured surface (sometimes "etched" by increasingthe temperature to about 100 C for several minutes to let
some ice sublime) is then shadowed with evaporated
platinum or gold at an average angle of 45 in a high vacuum
evaporator.
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A subclass of this is focused ion beam milling,
where gallium ions are used to produce an electron
transparent membrane in a specific region of the sample, for
example through a device within a microprocessor. Ion beam milling may also be used for cross-section polishing
prior to SEM analysis of materials that are difficult to prepare
using mechanical polishing.
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A second coat of carbon, evaporated perpendicular to the
average surface plane is often performed to improve stability
of the replica coating.
The specimen is returned to room temperature and pressure,then the extremely fragile "pre-shadowed" metal replica of
the fracture surface is released from the underlying biological
material by careful chemical digestion with acids, hypochlorite
solution or SDS detergent. Ion beam millingthins samples until they are transparent to
electrons by firing ions at the surface from an angle and
sputtering material from the surface.
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Conductive coating an ultrathin coating of electrically
conducting material, deposited either by high vacuum
evaporation or by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static electric
fields at the specimen due to the electron irradiation required
during imaging. The coating materials include gold,
gold/palladium, platinum, tungsten, graphite, etc.
Earthing to avoid electrical charge accumulation on a
conductive coated sample, it is usually electrically connected
to the metal sample holder. Often an electrically conductive
adhesive is used for this purpose.
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Transmission electron microscope The first TEM was built by Max Knoll and Ernst Ruska in 1931.
It is a microscopy technique in which a beam of electrons istransmitted through an ultra-thin specimen, interacting with
the specimen as it passes through.
An image is formed from the interaction of the electrons
transmitted through the specimen; the image is magnified
and focused onto an imaging device, such as
a fluorescent screen, on a layer of photographic film, or to be
detected by a sensor such as a CCD camera.
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TEMs are capable of imaging at a significantly
higher resolution than light microscopes, owing to the
small wavelength of electrons. At smaller magnifications TEM image contrast is due to
absorption of electrons in the material, due to the thickness
and composition of the material.
At higher magnifications complex wave interactions modulatethe intensity of the image, requiring expert analysis of
observed images.
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The first practical TEM,
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Source formation
TEM consists of an emission source, which may bea tungsten filament, or a lanthanum hexaboride source.
By connecting this gun to a high voltage source (typically 100300 kV) the gun will, given sufficient current, begin to emitelectrons either by thermionic or field electron emission intothe vacuum.
This extraction is usually aided by the use of a Wehneltcylinder.
Once extracted, the upper lenses of the TEM allow for theformation of the electron probe to the desired size andlocation for later interaction with the sample.
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Manipulation of the electron beam is performed using two
physical effects.
The interaction of electrons with a magnetic field will causeelectrons to move according to the right hand rule, thus
allowing for electromagnets to manipulate the electron beam.
The use of magnetic fields allows for the formation of a
magnetic lens of variable focusing power, the lens shape
originating due to the distribution of magnetic flux.
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Additionally, electrostatic fields can cause the electrons to be
deflected through a constant angle.
Coupling of two deflections in opposing directions with a
small intermediate gap allows for the formation of a shift inthe beam path, this being used in TEM for beam shifting,
subsequently this is extremely important to STEM.
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Optics
The lenses of a TEM allow for beam convergence, with
the angle of convergence as a variable parameter, givingthe TEM the ability to change magnification simply bymodifying the amount of current that flows through thecoil, quadrupole or hexapole lenses.
The quadrupole lens is an arrangement ofelectromagnetic coils at the vertices of the square,enabling the generation of a lensing magnetic fields, thehexapole configuration simply enhances the lenssymmetry by using six, rather than four coils.
Typically a TEM consists of three stages of lensing. The stages are the condensor lenses, the objective
lenses, and the projector lenses.
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The condensor lenses are responsible for primary beam
formation, whilst the objective lenses focus the beam that
comes through the sample itself .
The projector lenses are used to expand the beam onto the
phosphor screen or other imaging device, such as film.
The magnification of the TEM is due to the ratio of the
distances between the specimen and the objective lens'
image plane.
Additional quad or hexapole lenses allow for the correction of
asymmetrical beam distortions, known as astigmatism.
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Display Imaging systems in a TEM consist of a phosphor screen, which
may be made of fine (10100 m) particulate zinc sulphide,
for direct observation by the operator.
Optionally, an image recording system such as film based or
doped YAG screen coupled CCDs.
Typically these devices can be removed or inserted into the
beam path by the operator as required.
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Components
The electron source of the TEM is at the top, where thelensing system (4,7 and 8) focuses the beam on the specimen
and then projects it onto the viewing screen .The beamcontrol is on the right (13 and 14)
A TEM is composed of several components, which include avacuum system in which the electrons travel, an electronemission source for generation of the electron stream, a
series of electromagnetic lenses, as well as electrostaticplates.
The latter two allow the operator to guide and manipulate thebeam as required. Also required is a device to allow theinsertion into, motion within, and removal of specimens fromthe beam path.
Imaging devices are subsequently used to create an imagefrom the electrons that exit the system.
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Specimen stage
TEM specimen stage designs include airlocks to allow for
insertion of the specimen holder into the vacuum withminimal increase in pressure in other areas of the microscope.
The specimen holders are adapted to hold a standard size of
grid upon which the sample is placed or a standard size of
self-supporting specimen. Standard TEM grid sizes are a 3.05 mm diameter ring, with a
thickness and mesh size ranging from a few to 100 m.
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The sample is placed onto the inner meshed area having
diameter of approximately 2.5 mm.
Usual grid materials are copper, molybdenum, gold orplatinum. This grid is placed into the sample holder, which is
paired with the specimen stage.
A wide variety of designs of stages and holders exist,
depending upon the type of experiment being performed. Once inserted into a TEM, the sample often has to be
manipulated to present the region of interest to the beam,
such as in single grain diffraction, in a specific orientation.
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Each design must accommodate the matching holder to allow
for specimen insertion without either damaging delicate TEM
optics or allowing gas into TEM systems under vacuum.
The most common is the side entry holder, where the
specimen is placed near the tip of a long metal (brass or
stainless steel) rod, with the specimen placed flat in a small
bore.
Along the rod are several polymer vacuum rings to allow for
the formation of a vacuum seal of sufficient quality, when
inserted into the stage.
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To accommodate this, the TEM stage includes mechanisms for
the translation of the sample in the XY plane of the sample,
for Z height adjustment of the sample holder, and usually for
at least one rotation degree of freedom for the sample.
Thus a TEM stage may provide four degrees of freedom for
the motion of the specimen.
Most modern TEMs provide the ability for two orthogonal
rotation angles of movement with specialized holder designs
called double-tilt sample holders.
Two main designs for stages in a TEM exist, the side-entry and
top entry version.
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The stage is thus designed to accommodate the rod, placingthe sample either in between or near the objective lens,
dependent upon the objective design. When inserted into the stage, the side entry holder has its tip
contained within the TEM vacuum, and the base is presented
to atmosphere, the airlock formed by the vacuum rings.
Insertion procedures for side-entry TEM holders typicallyinvolve the rotation of the sample to trigger micro
switches that initiate evacuation of the airlock before the
sample is inserted into the TEM column.
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The second design is the top-entry holder consists of a
cartridge that is several cm long with a bore drilled down the
cartridge axis. The specimen is loaded into the bore, possibly
utilising a small screw ring to hold the sample in place. Thiscartridge is inserted into an airlock with the bore
perpendicular to the TEM optic axis.
When sealed, the airlock is manipulated to push the cartridge
such that the cartridge falls into place, where the bore holebecomes aligned with the beam axis, such that the beam
travels down the cartridge bore and into the specimen. Such
designs are typically unable to be tilted without blocking the
beam path or interfering with the objective lens.
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Electron gun
The electron gun is formed from several components: the
filament, a biasing circuit, a Wehnelt cap, and an extractionanode.
By connecting the filament to the negative component power
supply, electrons can be "pumped" from the electron gun to
the anode plate, and TEM column, thus completing thecircuit.
The gun is designed to create a beam of electrons exiting
from the assembly at some given angle, known as the gun
divergence semiangle, .
h h l l d h h h h h
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By constructing the Wehnelt cylinder such that it has a higher
negative charge than the filament itself, electrons that exit the
filament in a diverging manner are, under proper operation,
forced into a converging pattern the minimum size of which isthe gun crossover diameter.
The thermionic emission current density, J, can be related to
the work function of the emitting material and is a Boltzmann
distribution given below, whereAis a constant, is the work
function and T is the temperature of the material.
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This equation shows that in order to achieve sufficient current
density it is necessary to heat the emitter, taking care not to
cause damage by application of excessive heat, for this reason
materials with either a high melting point, such as tungsten,
or those with a low work function (LaB6) are required for the
gun filament.
Furthermore both lanthanum hexaboride and tungsten
thermionic sources must be heated in order to achieve
thermionic emission, this can be achieved by the use of a
small resistive strip.
To prevent thermal shock, there is often a delay enforced in
the application of current to the tip, to prevent thermalgradients from damaging the filament, the delay is usually a
few seconds for LaB6, and significantly lower for tungsten.
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Electron lens
Electron lenses are designed to act in a manner emulating
that of an optical lens, by focusing parallel rays at someconstant focal length.
Lenses may operate electrostatically or magnetically.
The majority of electron lenses for TEM
utilise electromagnetic coils to generate a convex lens.
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For these lenses the field produced for the lens must be
radially symmetric, as deviation from the radial symmetry of
the magnetic lens causes aberrations such as astigmatism,
and worsens spherical and chromatic aberration.
Electron lenses are manufactured from iron, iron-cobalt or
nickel cobalt alloys,such as permalloy.
These are selected for their magnetic properties, such
as magnetic saturation, hysteresis and permeability.
The exact dimensions of the gap, pole piece internal diameter
and taper, as well as the overall design of the lens is often
performed by finite element analysis of the magnetic field,
whilst considering the thermal and electrical constraints ofthe design.
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The coils which produce the magnetic field are located within
the lens yoke.
The coils can contain a variable current, but typically utilisehigh voltages, and therefore require significant insulation in
order to prevent short-circuiting the lens components.
Thermal distributors are placed to ensure the extraction of
the heat generated by the energy lost to resistance of the coil
windings.
The windings may be water-cooled, using a chilled water
supply in order to facilitate the removal of the high thermal
duty.
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Apertures
Apertures are annular metallic plates, through which
electrons that are further than a fixed distance from the opticaxis may be excluded.
These consist of a small metallic disc that is sufficiently thick
to prevent electrons from passing through the disc, whilst
permitting axial electrons. This permission of central electrons in a TEM causes two
effects simultaneously: firstly, apertures decrease the beam
intensity as electrons are filtered from the beam, which may
be desired in the case of beam sensitive samples.
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Secondly, this filtering removes electrons that are scattered to
high angles, which may be due to unwanted processes such as
spherical or chromatic aberration, or due to diffraction from
interaction within the sample.
Aperture assemblies are mechanical devices which allow for
the selection of different aperture sizes, which may be used
by the operator to trade off intensity and the filtering effect of
the aperture.
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Imaging methods
Imaging methods in TEM utilize the information contained in
the electron waves exiting from the sample to form an image.
The projector lenses allow for the correct positioning of this
electron wave distribution onto the viewing system.
The observed intensity of the image, I, assuming sufficiently
high quality of imaging device, can be approximated as
proportional to the time-averageamplitude of the electron
wavefunctions, where the wave which form the exit beam is
denoted by .
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From the previous equation, it can be deduced that the
observed image depends not only on the amplitude of beam,
but also on the phase of the electrons, although phase effects
may often be ignored at lower magnifications.
Higher resolution imaging requires thinner samples and
higher energies of incident electrons.
Therefore the sample can no longer be considered to be
absorbing electrons, via a Beer's law effect, rather the samplecan be modelled as an object that does not change the
amplitude of the incoming electron wavefunction.
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Rather the sample modifies the phase of the incoming wave;
this model is known as a pure phase object, for sufficiently
thin specimens phase effects dominate the image,
complicating analysis of the observed intensities. For example, to improve the contrast in the image the TEM
may be operated at a slight defocus to enhance contrast,
owing to convolution by the contrast transfer function of the
TEM, which would normally decrease contrast if the samplewas not a weak phase object.
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Contrast formation
Contrast formation in the TEM depends greatly on the mode
of operation. Complex imaging techniques, which utilise the unique ability
to change lens strength or to deactivate a lens, allow for many
operating modes.
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Bright field
The most common mode of operation for a TEM is the bright
field imaging mode.
In this mode the contrast formation, when considered
classically, is formed directly by occlusion and absorption of
electrons in the sample.
Thicker regions of the sample, or regions with a higher atomic
number will appear dark, whilst regions with no sample in the
beam path will appear brighthence the term "bright field".
The image is in effect assumed to be a simple two
dimensional projection of the sample down the optic axis, and
to a first approximation may be modelled via Beer's law,morecomplex analyses require the modelling of the sample to
include phase information.
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Diffraction contrast
Samples can exhibit diffraction contrast, whereby the electron
beam undergoes Bragg scattering, which in the case of acrystalline sample, disperses electrons into discrete locations
in the back focal plane.
By the placement of apertures in the back focal plane, i.e. the
objective aperture, the desired Bragg reflections can be
selected (or excluded), thus only parts of the sample that are
causing the electrons to scatter to the selected reflections will
end up projected onto the imaging apparatus.
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If the reflections that are selected do not include the
unscattered beam), then the image will appear dark wherever
no sample scattering to the selected peak is present, as such a
region without a specimen will appear dark. This is known as a
dark-field image.
Modern TEMs are often equipped with specimen holders that
allow the user to tilt the specimen to a range of angles in
order to obtain specific diffraction conditions, and aperturesplaced above the specimen allow the user to select electrons
that would otherwise be diffracted in a particular direction
from entering the specimen.
Sample preparation
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Sample preparation
Sample preparation in TEM can be a complex procedure. TEM
specimens are required to be at most hundreds ofnanometers thick, as unlike neutron or X-Ray radiation the
electron beam interacts readily with the sample, an effect
that increases roughly with atomic number squared .
High quality samples will have a thickness that is comparable
to the mean free path of the electrons that travel through the
samples, which may be only a few tens of nanometers.
Preparation of TEM specimens is specific to the material
under analysis and the desired information to obtain from the
specimen.
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Materials that have dimensions small enough to be electron
transparent, such as powders or nanotubes, can be quickly
prepared by the deposition of a dilute sample containing the
specimen onto support grids or films.
In the biological sciences in order to withstand the
instrument vacuum and facilitate handling, biological
specimens can be fixated using either a negative
staining material such as uranyl acetate or by plasticembedding.
Alternately samples may be held at liquid
nitrogen temperatures after embedding in vitreous ice.
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Tissue sectioning
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Tissue sectioning
By passing samples over a glass or diamond edge, small, thin
sections can be readily obtained using a semi-automatedmethod.
This method is used to obtain thin, minimally deformed
samples that allow for the observation of tissue samples.
Additionally inorganic samples have been studied, such asaluminium, although this usage is limited owing to the heavy
damage induced in the less soft samples.
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To prevent charge build-up at the sample surface, tissue
samples need to be coated with a thin layer of conducting
material, such as carbon, where the coating thickness is
several nanometers.
This may be achieved via an electric arc deposition process
using a sputter coating device.
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Sample staining
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Sample staining
Details in light microscope samples can be enhanced
by stains that absorb light; similarly TEM samples of biologicaltissues can utilize high atomic number stains to enhance
contrast.
The stain absorbs electrons or scatters part of the electron
beam which otherwise is projected onto the imaging system.
Compounds ofheavy metals such
as osmium, lead, uranium or gold (in immunogold labelling)
may be used prior to TEM observation to selectively deposit
electron dense atoms in or on the sample in desired cellular
or protein regions, requiring an understanding of how heavymetals bind to biological tissues.
Mechanical milling
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Mechanical milling
Mechanical polishing may be used to prepare samples.
Polishing needs to be done to a high quality, to ensureconstant sample thickness across the region of interest.
A diamond, or cubic boron nitride polishing compound may
be used in the final stages of polishing to remove any
scratches that may cause contrast fluctuations due to varying
sample thickness.
Even after careful mechanical milling, additional fine
methods such as ion etching may be required to perform final
stage thinning.
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Chemical etching
Certain samples may be prepared by chemical etching,
particularly metallic specimens.
These samples are thinned using a chemical etchant, such as
an acid, to prepare the sample for TEM observation.
Devices to control the thinning process may allow the
operator to control either the voltage or current passing
through the specimen, and may include systems to detect
when the sample has been thinned to a sufficient level of
optical transparency.
Ion etching
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Ion etching
Ion etching is a sputtering process that can remove very fine
quantities of material.
Ion etching uses an inert gas passed through an electric field
to generate a plasma stream that is directed to the sample
surface.
Acceleration energies for gases such as argon are typically a
few kilovolts. The sample may be rotated to promote even
polishing of the sample surface.
The sputtering rate of such methods is on the order of tens of
micrometers per hour, limiting the method to only extremely
fine polishing.
l f d b h d h b d
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More recently focused ion beam methods have been used to
prepare samples. FIB is a relatively new technique to prepare
thin samples for TEM examination from larger specimens.
Because FIB can be used to micro-machine samples very
precisely, it is possible to mill very thin membranes from a
specific area of interest in a sample, such as a semiconductor
or metal.
Replication
Samples may also be replicated using cellulose acetate film,
the film subsequently coated with a heavy metal, the original
film melted away, and the replica imaged on the TEM. This
technique is used for both materials and biological samples.
Tissue preparation for transmission
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Tissue preparation for transmission
electron microscopy
The fundamental principle underlying TEM is that electrons
pass through the section to give an image of the specimen.
However, the electron beam is only capable of penetrating
around 100 nm, so, to obtain a high-quality image and
optimize the resolution of the instrument, it is necessary tosection the tissue to a thickness of around 80 nm.
Sectioning at this level requires tissues to be embedded in
extremely rigid material.
Clearly the wax embedding media used in light microscopy
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y g g py
are not suitable.
In routine TEM synthetic embedding resins are used which are
capable of withstanding the vacuum in the electronmicroscope column and the heat generated as the electrons
pass through the section.
Hydrophobic epoxy resins are preferred.
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Specimen handling
In order to preserve the ultrastructure of the cell it is crucial
that samples are fixed as soon as possible after the biopsy is
taken.
The standard approach is to immerse the specimen in fixative
(preferably pre-cooled to 4C) immediately after collection.
Once in fixative, the specimen is cut into smaller samples
using a scalpel or razor blade.
At thi i t th ti h ld b i t t d d di t d t
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At this point the tissue should be orientated and dissected to
optimize exposure of the critical diagnostic features during
sectioning and screening.
Dissection must also facilitate the penetration of fixatives and
processing reagents.
The final tissue blocks may be in the form of thin sheets or
small cubes (~1 mm3), although the risk of sampling error
increases as the sample size decreases.
In general, the volume of fixative should be at least 10 times
the volume of the tissue.
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It is also vital to ensure that the tissue remains completely
submerged in the fixative small pieces may adhere to the
inside of the lid of the biopsy container.
It should also be noted that fixatives and processing reagents
penetrate different tissues at different rates, and some tissues
(such as liver) very poorly.
Needle biopsies of liver may need to be cut longitudinally to
ensure adequate fixation.
If a delay in fixation is unavoidable, damage can be minimized
by holding the tissue (for a short time only) in chilled normal
saline.
However, the tissue must not be frozen at any point.
Fixation
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The fixatives used in TEM generally comprise a fixing agent in
buffer (to maintain pH) and, if necessary, with various
additives to control osmolarity and ionic composition.
Other factors that affect fixation include fixative concentration
and temperature, and the duration of fixation.
The standard protocol involves primary fixation with an
aldehyde (usually glutaraldehyde) to stabilize proteins,followed by secondary fixation in osmium tetroxide to retain
lipids.
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Fixative concentration
Glutaraldehyde is effective at a concentration of between
1.5% and 4%, with 2.5% the simplest to prepare from the 25%stock solutions available commercially.
Osmium tetroxide is usually used at a concentration of 1% or
2%.
Temperature
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Temperature
Fixation at room temperature improves the penetration rate
(particularly of aldehyde fixatives) and reduces the time
required for fixation.
Although it also increases the risk of autolytic change.
Osmium tetroxide is generally used at room temperature.
Duration of fixation
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The time required for optimal fixation depends on a range of
factors, including the type of tissue, the size of the sample,
and the type of fixative and buffer system used. In most circumstances immersion of 0.51.0 mm 3 blocks of
tissue in 2.5% glutaraldehyde fixative for 26 hours is
sufficient.
Secondary fixation in 1% osmium tetroxide for 6090 minutesis usually effective.
much longer times are required if osmium tetroxide is the
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much longer times are required if osmium tetroxide is the
primary fixative.
The use of microwave irradiation can accelerate fixation times
in aldehyde fixative to as little as 510 seconds (Leong1994),
after which the sample may be stored in buffer or processed
immediately.
Buffers
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Fixatives are normally buffered within the range of pH 7.2
7.6.
osmolarity and ionic composition of the buffer should mimicthat of the tissue being fixed.
Non-ionic molecules such as glucose, sucrose or dextran are
used to adjust tonicity as these will not influence the ionic
constitution of the buffer. The addition of various salts,particularly calcium and
magnesium, is thought to improve tissue preservation,
possibly by stabilizing membranes.
Aldehyde fixatives
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Aldehyde fixatives Glutaraldehyde
Glutaraldehyde is the most widely used primary fixative in
TEM.
The most important reaction of glutaraldehyde, that of
stabilizing proteins, is thought to occur via a cross-linking
mechanism involving the amino groups of lysine and other
amino acids through the formation of pyridine intermediaries.
Lipids and most phospholipids are not fixed and will be
extracted during subsequent processing without secondary
fixation.
Phosphate buffers
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Phosphate buffers (Gomori 1955) have the disadvantage of
being good growth media for molds and other
microorganisms.
Additionally, most metal ions form insoluble phosphates,
which restricts the use of this buffer (the phosphates of
sodium, potassium and ammonium are soluble).
Nevertheless, phosphate buffers are the buffer of choice as
they are non-toxic and work well with most tissues.
F ld h d
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Formaldehyde
Formaldehyde that has been freshly prepared from
paraformaldehyde powder is adequate for TEM as it lacks
impurities and also has the advantage of a faster penetration
rate compared
with glutaraldehyde.
Paraformaldehyde is often recommended in electron
immunocytochemistry as epitopes are less likely to be
significantly altered during fixation and, if required, antigen
unmasking is more effective.
Osmium tetroxide
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The use of osmium tetroxide fixation to preserve lipids is
fundamental to electron microscopy.
While primary fixation in osmium tetroxide is effective, its
extremely slow penetration rate can give rise to autolytic
changes.
For this reason osmium tetroxide is almost always used as a
secondary fixative (termed postfixation) after primary
fixation in aldehyde.
The penetration rate of osmium tetroxide is also higher in
stabilized tissue, such that immersion for 6090 minutes is
usually sufficient for most specimens.
Osmi m tetro ide is s all s pplied in cr stalline form sealed
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Osmium tetroxide is usually supplied in crystalline form sealed
in glass ampoules.
Extreme care should be exercised when preparing this
material and gloves and eye protection should always be
worn.
Osmium tetroxide can be prepared as an aqueous solution,
although it can also be made in the same buffer used to
prepare the primary fixative.
Wash buffer and staining
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g
Material that is to be retained may be rinsed briefly (in a
buffer compatible with the fixative vehicle), then stored in
fresh buffer.
Tissue that is for immediate processing should be washed in
buffer before post-fixation in osmium tetroxide, then washed
again in buffer or water to remove excess osmium.
This is critical as osmium tetroxide and alcohol react to form a
black precipitate.
An optional step at this point is to immerse tissues after post-
fixation in 2% aqueous uranyl acetate.
This en bloc staining procedure adds to the contrast of the
final sections and improves preservation.
Dehydration
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y The most common embedding compounds usednin TEM are
epoxy resins.
These are totally immiscible with water, thus requiringspecimens to be dehydrated.
Dehydration is performed by passing the specimen through
increasing concentrations of an organic solvent.
The most frequently used dehydrants are acetone and
ethanol.
Acetone should be avoided if en bloc staining with uranyl
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Acetone should be avoided if en bloc staining with uranyl
acetate has been performed to prevent precipitation of
uranium salts.
Ethanol overcomes this difficulty but requires the use of
propylene oxide (1,2-epoxypropane) as a transition solvent to
facilitate resin infiltration.
Residual dehydrant can result in soft or patchy blocks.
Embedding
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g The standard practice following dehydration and, ifnrequired,
treatment with a transitional solvent, is toninfiltrate the tissue
sample with liquid resin. This usually requires gradual introduction of the
resin,beginning with a 50 : 50 mix of transition solvent
(propylene oxide) and resin followed by a 25 : 75 transition
solvent resin mix, then, finally, pure resin. An hour in each of the preliminary infiltration steps is usually
adequate, although it is preferable to leave samples in pure
resin for 24 hours.
Gentle agitation using a low-speed, angled rotator during
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Gentle agitation using a low speed, angled rotator during
these steps is recommended, as failure to completely infiltrate
the tissue will cause major sectioning difficulties.
Once infiltrated, tissue samples are placed in an appropriatemold which is filled with resin and allowed to polymerize
using heat.
A paper strip bearing the tissue identification code written in
pencil or laser-printed is included.
Capsules made from polyethylene are recommended as they
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Capsules made from polyethylene are recommended as they
do not react with resin, as are flat embedding molds made of
silicone rubber.
Polymerized blocks can be easily removed from the latter bybending the mold, which can then be reused.
Polyethylene capsules can be cut away from the block using a
razor or scalpel blade or the block can be extruded from the
capsule using large forceps or pliers.
Epoxy resins
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These resins contain a characteristic chemical group in which
an oxygen and two carbon atoms bond to form a three
membered ring (epoxide). Cross-linking between these groups creates a three-
dimensional polymer of great mechanical strength.
The polymerization process generates very little shrinkage
(usually less than 2%) and, once complete, is permanent.
A ll th i ti f if l i ti d l
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As well as their properties of uniform polymerization and low
shrinkage, epoxy resins also preserve tissue ultrastructure, are
stable in the electron beam, section easily and are readily
available.
Epoxy resins usually comprise four ingredients: the
monomeric resin, a hardener, an accelerator and a plasticizer.
Although manufacturers provide advice on the appropriate
proportions, the hardness and flexibility of blocks and pthe
individual components.olymerization times can be
manipulated by varying the amount of the individual
components.
Acrylic resins
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Acrylic resins can rapidly infiltrate fixed, dehydrated tissues at
room temperature.
Currently available acrylics are now polymerized using a cross-linking process, thereby overcoming earlier disadvantages.
Acrylic monomers are of low viscosity, and both hydrophilic
and hydrophobic forms are obtainable.
Acrylic resins react by free radical polymerization, which can
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y y p y ,
be initiated using light,heat or a chemical accelerator
(catalyst) at room temperature.
The main commercial acrylic resins are LR White
and LR Gold and the Lowicryl series.
Tissue processing schedules
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Manual tissue processing is best performed by keeping the
tissue sample in the same vial throughout, and using a fine
pipette to change solutions. When processing multiple samples, take care not to cross-
contaminate specimensuse separate pipettes.
All vials must be clearly labeled and labels must be solvent-
proof. It is advantageous to agitate tissue specimensthroughout the processing cycle to enhance reagent
permeation.
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Cell suspensions or particulate matter
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Cell suspensions (such as fine needle biopsy aspirates, bone
marrow specimens or cytology samples) or particulate
materials (including fluid aspirates, tissue fragments orproducts and specimens for the assessment of ciliary
structures) are best embedded in a protein support medium
before processing.
Plasma, agar or bovine serum albumen (BSA) can be used.
The addition of tannic acid during the preparation of ciliary
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specimens gives improved visualization of axonemal
components.
The tannic acid is thought to act as a fixative and also amordant, facilitating the binding of heavy metal stains.
Double en bloc staining with uranyl acetate and lead
aspartate may also improve the visibility of dynein arms.
Ultramicrotomy
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Glass knives
Knives are prepared from commercially available plate glass
strips manufactured specifically for ultramicrotomy.
Before use, the strips should be washed thoroughly with
detergent, then rinsed in distilled water and alcohol and dried
using lint-free paper.
Higher angle knives (up to 55) are best suited to cutting hard
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Higher angle knives (up to 55 ) are best suited to cutting hard
materials, while softer blocks respond better to shallower
(35) angle knives.
In ultramicrotomy, thin sections are floated out for collection
as they are cut.
This requires a small trough to be attached directly to the
knife.
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Diamond knives A well maintained diamond knife is capable of cutting any
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A well-maintained diamond knife is capable of cutting any
type of resin block and most biological and many non-
biological materials.
Diamond knives are brittle but very durable and will continue
to cut for quite some time provided they are kept clean and
treated carefully.
The cutting edge can be cleaned by carefully running a
polystyrene cleaning strip along the edge.
A diamond knife must only be used to cut ultra-thin sections
and should never be used drywithout a trough fluid.
Trough fluidsh l d bl fl d l d
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The simplest and most suitable fluid routinely used in section
collecting troughs is distilled or deionized water.
It is important to ensure that the correct level of fluid isadded.
If the level is too high, the fluid will be drawn over the cutting.
If the level is too low, sections will accumulate on the cutting
edgeand will not float out.
Block trimming
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Once polymerized, blocks must be cleared of excess resin to
expose the tissue for sectioning.
At the completion of this process the trimmed area shouldresemble a flat-topped pyramid with a square or trapezium-
shaped face.
Trimming the block can be achieved manually or by using the
ultramicrotome.
At its simplest, manualtrimming can be performed by
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p , g p y
mounting the block in a suitable holder under a dissecting
microscope and removing the surplus resin with a single-
edged razor blade.
Alternatively, the block is positioned in the ultramicrotome
and mechanically trimmed using a glass knife.
Collection of sectionsUl hi i d i id f
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Ultra-thin sections are mounted onto specimen grids for
viewing.
Grids measure 3.05 mm in diameter and are made ofconductive material, commonly copper, nickel or gold,
although silver, palladium, molybdenum, aluminum, titanium,
stainless steel, nylon-carbon and combination varieties are
available.
A large range of patterns and mesh sizes are available with
200 square mesh being commonly used, although slotted,
parallel bar and hexagonal patterns are also standard.
Staining
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Tissues are stained at several points during preparation
1. During secondary fixation
2. When uranyl acetate is used during the post-fixation wash.
3. By staining the sections with lead and uranium Salts.
The standard method for staining sections is to float the grids,
section-side-down, on drops of staining solution for the
required time.
After each staining step, the grid is washed under a gentle
stream of distilled water or by dipping in distilled water.
Finally, the grids are dried using clean lint free filter paper.
The use of TEM for diagnostics
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TEM is used to obtain structural and compositional
information that cannot be acquired realistically using an
alternative technique. In practice, TEM is rarely used alone and is generally part of
an integrated diagnostic protocol.
The Scanning Electron Microscope
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It is a type of electron microscope that produces images of a
sample by scanning it with a focused beam of electrons.
The electrons interact with atoms in the sample, producing
various signals that can be detected and that contain
information about the sample's surface topography and
composition.
The electron beam is generally scanned in a rasterscan pattern, and the beam's position is combined with the
detected signal to produce an image.
SEM can achieve resolution better than 1 nanometer.
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The most common mode of detection is by secondary
electrons emitted by atoms excited by the electron beam.
By scanning the sample and detecting the secondary
electrons, an image displaying the tilt of the surface is
created.
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SEM opened sample chamber
Principles and capacities
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The types of signals produced by a SEM include secondary
electrons , back-scattered electrons current and transmittedelectrons.
Secondary electron detectors are standard equipment in all
SEMs, but it is rare that a single machine would have
detectors for all possible signals. The signals result from interactions of the electron beam with
atoms at or near the surface of the sample.
In the most common or standard detection mode, secondary
electron imaging, the SEM can produce very high-resolution
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electron imaging, the SEM can produce very high resolution
images of a sample surface, revealing details less than 1 nm in
size.
Due to the very narrow electron beam, SEM micrographs
have a large depth of field yielding a characteristic three-
dimensional appearance useful for understanding the surface
structure of a sample.
Sample preparation
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For conventional imaging in the SEM, specimens must
be electrically conductive, at least at the surface,
and electrically grounded to prevent the accumulationof electrostatic charge at the surface.
Nonconductive specimens tend to charge when scanned by
the electron beam, and especially in secondary electron
imaging mode, this causes scanning faults and other imageartifacts.
They are therefore usually coated with an ultrathin coating of
electrically conducting material, deposited on the sample
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electrically conducting material, deposited on the sample
either by low-vacuum sputter coating or by high-vacuum
evaporation.
Conductive materials in current use for specimen coating
include gold,
gold/palladium alloy, platinum, osmium, iridium, tungsten, chr
omium, and graphite.
Biological samples
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For SEM, a specimen is normally required to be completely
dry, since the specimen chamber is at high vacuum.
Soft-bodied organisms usually require chemical fixation to
preserve and stabilize their structure.
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, and replacement of these solvents in
turn with a transitional fluid such as liquid carbon
dioxide by critical point drying.
If the SEM is equipped with a cold stage for cryo
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microscopy, cryofixation may be used and low-temperature
scanning electron microscopy performed on the cryogenically
fixed specimens.
Cryo-fixed specimens may be cryo-fractured under vacuum in
a special apparatus to reveal internal structure, sputter-
coated, and transferred onto the SEM cryo-stage while still
frozen.
Scanning process and image formation
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In a typical SEM, an electron beam is thermionically emitted
from an electron gun fitted with a tungsten filament cathode.
Tungsten is normally used in thermionic electron guns
because it has the highest melting point and lowest vapour
pressure of all metals, thereby allowing it to be heated for
electron emission.
The electron beam, which typically has an energy ranging
from 0.2 keV to 40 keV, is focused by one or two condenser
lenses to a spot about 0.4 nm to 5 nm in diameter.
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Magnification
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Magnification in a SEM can be controlled over a range of up to
6 orders of magnitude from about 10 to 500,000 times.
Unlike optical and transmission electron microscopes, image
magnification in the SEM is not a function of the power of
the objective lens.
SEMs may have condenser and objective lenses, but their
function is to focus the beam to a spot, and not to image the
specimen.
Provided the electron gun can generate a beam with
sufficiently small diameter, a SEM could in principle work
entirely without condenser or objective lenses, although it
might not be very versatile or achieve very high resolution.
In a SEM, magnification results from the ratio of the
dimensions of the raster on the specimen and the raster on
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dimensions of the raster on the specimen and the raster on
the display device.
Assuming that the display screen has a fixed size, highermagnification results from reducing the size of the raster on
the specimen, and vice versa.
Magnification is therefore controlled by the current supplied
to the x, y scanning coils, or the voltage supplied to the x, ydeflector plates, and not by objective lens power.
Color The most common configuration for an SEM produces a single
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The most common configuration for an SEM produces a single
value per pixel, with the results usually rendered as black-
and-white images.
However, often these images are then colorized through the
use of feature-detection software, or simply by hand-editing
using a graphics editor.
This is usually for aesthetic effect or for clarifying structure,and generally does not add information about the specimen.
A combination of backscattered and secondary electron
signals can be assigned to different colors and superimposed
on a single color micrograph displaying simultaneously theproperties of the specimen.
Resolution of the SEM
Th i l l i f h SEM d d h i f h
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The spatial resolution of the SEM depends on the size of theelectron spot, which in turn depends on both the wavelength
of the electrons and the electron-optical system that producesthe scanning beam.
The resolution is also limited by the size of the interactionvolume, or the extent to which the material interacts with theelectron beam.
The SEM has compensating advantages, though, including theability to image a comparatively large area of the specimen;the ability to image bulk materials (not just thin films or foils);and the variety of analytical modes available for measuringthe composition and properties of the specimen.
Depending on the instrument, the resolution can fallsomewhere between less than 1 nm and 20 nm. By 2009, Theworld's highest SEM resolution at high-beam energies (0.4 nmat 30 kV) is obtained with the Hitachi SU-9000.
Interference microscopy
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It is an optical microscopy illumination technique used to
enhance the contrast in unstained, transparent samples.
It works on the principle of interferometry to gain information
about the optical path length of the sample, to see otherwise
invisible features.
A relatively complex lighting scheme produces an image with
the object appearing black to white on a grey background.
This image is similar to that obtained by phase contrast
microscopy but without the bright diffraction halo.
DIC works by separating a polarized light source into two
orthogonall polari ed m t all coherent parts hich are
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orthogonally polarized mutually coherent parts which are
spatially displaced (sheared) at the sample plane, and
recombined before observation. The interference of the two parts at recombination is sensitive
to their optical path difference. Adding an adjustable offset
phase determining the interference at zero optical path
difference in the sample, the contrast is proportional to thepath length gradient along the shear direction, giving the
appearance of a three-dimensional physical relief
corresponding to the variation of optical density of the
sample, emphasising lines and edges though not providing a
topographically accurate image.
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The light path
Th t f th b i diff ti l i t f t t
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The components of the basic differential interference contrastmicroscope setup.
1. Unpolarised light enters the microscope and is polarised at45.
Polarised light is required for the technique to work.
2. The polarised light enters the first Nomarski-
modified Wollaston prism and is separated into two rayspolarised at 90 to each other, the sampling and reference
rays.
Wollaston prisms are a type of prism made of two layers of acrystalline substance, such as quartz, which, due to the
variation of refractive index depending on the polarisation ofthe light, splits the light according to its polarisation.
The Nomarski prism causes the two rays to come to a focal
point outside the body of the prism, and so allows greater
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point outside the body of the prism, and so allows greater
flexibility when setting up the microscope, as the prism can be
actively focused. 3. The two rays are focused by the condenser for passage
through the sample.
These two rays are focused so they will pass through two
adjacent points in the sample, around 0.2 mapart. The sample is effectively illuminated by two coherent light
sources, one with 0 polarisation and the other with 90
polarisation. These two illuminations are, however, not quite
aligned, with one lying slightly offset with respect to theother.
The rays travel through adjacent areas of the sample,
separated by the shear. The separation is normally similar to
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p y p y
the resolution of the microscope.
They will experience different optical path lengths where theareas differ in refractive index or thickness.
This causes a change in phase of one ray relative to the other
due to the delay experienced by the wave in the more optically
dense material.
The passage of many pairs of rays through pairs of adjacent
points in the sample means an image of the sample will now
be carried by both the 0 and 90 polarised light.
The light also carries information about the image invisible tothe human eye, the phase of the light. This is vital later.
This prism overlays the two bright field images and aligns
their polarisations so they can interfere. However, the images
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their polarisations so they can interfere. However, the images
do not quite line up because of the offset in illumination - this
means that instead of interference occurring between 2 raysof light that passed through the same point in the specimen,
interference occurs between rays of light that went
through adjacent points which therefore have a slightly
different phase.
Because the difference in phase is due to the difference in
optical path length, this recombination of light causes
"optical differentiation" of the optical path length, generating
the image seen.
The different polarisations prevent interference between
these two images at this point.
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these two images at this point.
5. The rays travel through the objective lens and are focused
for the second Nomarski-modified Wollaston prism. 6. The second prism recombines the two rays into one
polarised at 135. The combination of the rays leads
to interference, brightening or darkening the image at that
point according to the optical path difference.
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In the interference microscope the retarded rays are entirely
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In the interference microscope the retarded rays are entirely
separated from the direct or reference rays allowing improved
image contrast, color graduation, and quantitativemeasurements of phase change refractive index, dry mass of
cells , and section thickness.
Whenever light passes across the edge of an opaque object
the rays close to that edge are diffracted, or bent away fromtheir normal path.
If instead of a single edge, the rays pass through a narrow slit,
then the rays at the edge of the beam will fan out on either
side to quite wide angles.
Two slits closely side by side form two fans of rays which will
cross and if coherent will observably interfere
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cross and, if coherent,will observably interfere .
If each ray is regarded as a wave it can be seen that phase
conditions of increased amplitude and extinction are boundto occur at points where the waves cross and interfere.
The result of this in the microscope is a series of parallel
bands, alternately bright and dark across the field of view.
With white light, bands of the spectral colors are seen,
because the wavelengths making up white light are diffracted
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because the wavelengths making up white light are diffracted
at different angles.
With monochromatic light, the bands are alternately dark
and light, and of a single color.
The same effect can be shown if separate beams of coherent
light are reunited.
This phenomenon is known as interference.
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Two types of double-beam system have been used.
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One involved focusing the reference beam below the object
the double focus system and the other involved a lateral
displacement of the reference beam called shearing, where
the separation of the beams is very small.
The first birefringent prism in the condenser separates the
beams and after passing through the object they are
recombined by the second identical prism at the back of the
objective.
A different pair of prisms is required for each magnification.
This produces interference contrast and together with
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This produces interference contrast and together with
rotation of the polarizers enhances the three-dimensional
(3D) effect in the image.
This system is referred to as differential interference contrast
or DIC.
Additionally, only one such prism is required at the objective
level for all magnifications.
This system permits enhanced visualization of
immunohistochemical preparations.
(a) A Wollaston prism is so constructed
that rays passing through the center
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are in phase.
Those passing at other points have
a phase difference. The arrows anddot represent the optic axes of the
prisms, being at right angles to each
other.
(b) Ray path in the microscope. Each
ray is polarized on separation andthey vibrate at right angles to each
other, producing
interference colors when recombined.
Image The image is generated from two identical bright field images
being overlaid slightly offset from each other (typically around
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being overlaid slightly offset from each other (typically around
0.2 m), and the subsequent interference due to phase
difference converting changes in phase (and so optical path
length) to a visible change in darkness.
This interference may be either constructive or destructive,
giving rise to the characteristic appearance of three
dimensions.
The typical phase difference giving rise to the interference is
very small, very rarely being larger than 90 .
The image can be approximated as the differential of optical
path length with respect to position across the sample along
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path length with respect to position across the sample along
the shear, and so the differential of the refractive index
(optical density) of the sample.
The contrast can be adjusted using the offset phase, either by
translating the objective Nomarski prism, or by a lambda/4
waveplate between polarizer and the condenser Normarski
prism The resulting contrast is going from dark-field for zerophase offset to the typical relief seen for phase of ~590
degrees, to optical staining at 360 degrees, where the
extinguished wavelength shifts with the phase differential.
One non-biological area where DIC is useful is in the analysis
of planar silicon semiconductor processing. The thin (typically
100 1000 ) fil i ili i f l
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100-1000 nm) films in silicon processing are often mostly
transparent to visible light (e.g., silicon dioxide, silicon nitride
and polycrystalline silicon), and defects in them or
contamination lying on top of them become more visible. This
also enables the determination of whether a feature is a pit in
the substrate material or a blob of foreign material on top.
Etched crystalline features gain a particularly strikingappearance under DIC.
Image quality, when used under suitable conditions, is
outstanding in resolution and almost entirely free of artifacts
unlike phase contrast.
Advantages and disadvantages
DIC has strong advantages in uses involving live and unstained
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g g g
biological samples, such as a smear from a tissue culture or
individual water borne single-celled organisms. Its resolution and clarity in conditions such as this are
unrivaled among standard optical microscopy techniques.
The main limitation of DIC is its requirement for a transparent
sample of fairly similar refractive index to its surroundings. DIC is unsuitable for thick samples, such as tissue slices, and
highly pigmented cells. DIC is also unsuitable for most non
biological uses because of its dependence on polarisation,
which many physical samples would affect.
Polarized light microscopy 1stintroduced in 19thcent.
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Polarized light is a contrast enhancing technique that
improves the quality of the image obtained withbirefringent materials.
Substances or crystals capable of producing plane polarized
light are called birefringent.
This type of microscope differs from the normal one by usinga polarized light, in which the light waves vibrate in one
direction.
Its used in anisotropic materials (like minerals) because of
their birefringent optical properties they have several
refractive indices.
The Polarizing Lens Microscope
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When studying a specimen the light has to pass through a
polarizer (polarizing filter) and then in some cases through an
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analyzerto increase the quality of image contrast.
A polarizer is a filter that only allows specific light waves orvibrations to pass through it and focus them in a single plane.
An analyzer, mainly used as a second polarizer located above
the sample, determines the quantity and the direction of the
light that illuminates a sample. By changing the relationship of the polarizer and the analyzer,
its possible to determine the amount of absorbance,
reflection and refraction of the light through the microscope.
Analyzer(upper polarizer) -- a polarizingprism located above the microscope stage,
between the objective lens and the
eyepiece. This restricts the transmission of
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light vibrating perpendicular to the
polarizer. The analyzer can be slipped in or
out of the light path or rotated for partiallycrossed polarized light. Light passing
through the polarizer will not pass through
the analyzer unless the vibration direction
of the light is changed between the two
prisms. Anisotropic minerals can perform
this deed.
Polarizer(lower polarizer) -- a polarizing
prism located beneath the microscope
stage (between the light source and the
object of study). This restricts transmission
of light to that vibrating in only one (N-S)
direction. Some microscopes have a
different orientation direction. In effect, it
plane polarizes the incident light beam.
The use of polarized light in microscopy has manyuseful and diagnostic applications.
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With polarizing microscopy it is possible to determine
the color absorption, structure, composition andrefraction of light in isotropic (gases and liquids onerefractive index) and anisotropic substances.
Numerous crystals, fibrous structures pigments, lipids,
proteins, bone, and amyloid deposits exhibitbirefringence.
polarized light vibrates in only one plane, and can beproduced for microscopy purpose by passing natural
light through a polarizer, which is an optical componentmade from a substance that will allow vibrations ofonly one vibration direction to pass.
Optical birefringence
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Birefringence is formally defined as the double refraction of
light in a transparent, molecularly ordered material, which is
manifested by the existence of orientation-dependent
differences in refractive index.
How is light polarized and how does this help us identify specific minerals?
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1stpolarizer is oriented in vertically to the incident beam so itwill pass only the waves having vertical electrical field vectors.
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The waves passing through the 1st polarizer is subsequently
blocked by the 2nd
polarizer, since 2nd
polarizer is oriented athorizontal with respect to the electric field vectors in the lightwaves.
The concept of using two polarizer oriented at right angle withrespect to each other is called Cross polarization& is
fundamental concept of polarized light microscope.
Light entering a birefringent crystal such as calcite is split into two light
paths, each determined by a different refractive index (RI) and each
vibrating in one direction only (i.e. polarized) but at right angles to each
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other.
The higher the RI, the greater the retardation of the ray, so that each rayleaves the crystal at a different velocity. The high RI ray is called slow and
the low RI ray is calledfast.
There is also a phase difference between the rays, so that, if
they are recombined, interference occurs and various spectral
l
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colors are seen.
There will be a direction within a birefringent crystal alongwhich light may pass unaltered; this is called the optic axis.
Substances through which light can pass in any direction and
at the same velocity are called isotropic and are not able to
produce polarized light. Some substances and crystals can produce plane polarized
light by differential absorption and give rise to the
phenomenon of dichroism.
The dedicated polarizing microscope uses two polarizers. One,
always referred to as the polarizer, is placed beneath the
b t d d h ld i t t bl d t d t
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substage condenser and held in a rotatable graduated mount,
and can be removed from the light path when not required. The other, called the analyzer, is placed between the objective
and the eyepiece and is also graduated for measurement to
be taken.
A circular rotating stage would also be present for rotation ofthe specimen.
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(a) When polarizer and analyzer are parallel, rays vibrating in the parallel plane are
able to pass.
(b) When polarizer and analyzer are crossed, rays able to pass the polarizer are
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blocked by the analyzer. The condition when no light reaches the observer is
known as extinction.
The human eye is not able to distinguish any difference
between polarized and natural light, although when looking
through a single polarizer there is an obvious loss of intensity
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through a single polarizer there is an obvious loss of intensity,
some of which is due to the color of the filter, as well as thesplitting and absorption of the rays.
Polarizing spectacles used as sunglasses make full use of both
properties, but their chief advantage is the elimination of
glare and reflected light from such surfaces as water and
glass, which act as polarizers.
Looking through two polarizers, if their vibration directions
are parallel, results in a further slight loss of intensity due to
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the increase of the thickness and subsequent absorption, but
as one is rotated in relation to the other, intensity decreasesto extinction when the vibration directions are crossed, and at
right angles.
The first polarizer only allows the passage of rays vibrating in
its ownnvibration direction; if parallel, the second polarizerwill allow those rays to pass; if crossed, passage of the rays is
blocked.
Two phenomena detected in polarized light are interesting to
the histologist: birefringence and dichroism.
When a birefringent substance is rotated between two
crossed polarizers, the image appears and disappears
alternately at each 45 of rotation
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alternately at each 45 of rotation.
Hence, in a complete revolution of 360 the image appearsfour times, and likewise, it is extinguished completely four
times.
When one of the planes of vibration of the object is in a
parallel plane to the polarizer, only one part ray can develop,and its further passage is blocked by the analyzer in the
crossed position.
At 45, however, phase differences between the two rays
which can develop are able to combine in the analyzer and
form a visible image.
When a birefringent substance is rotated between crossed
polarizers, it is visible when it is in the diagonal position .
E ti ti h f it l f ib ti i ll l
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Extinction occurs when one of its planes of vibration is parallel
to either polarizer.
The color changes in a rotation of 90, and back to its original
color in the next 90
This is due to differential absorption of light depending upon
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This is due to differential absorption of light,depending upon
the vibration direction of the two rays in a birefringent
substance.
Weak birefringence in biological specimens is enhanced by
the addition of dyes or impregnating metals, in an orderly
linear alignment.
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A Acellular cementum B Cellular cementum X 50
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A- Acellular cementum B- Cellular cementum X 50