Specialised Microscope

8/10/2019 Specialised Microscope

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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, .

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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

Specialised Microscope

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