ELECTRON MICROSCOPY

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ELECTRON MICROSCOPY

• Ganesh Prasad Acharya
• Cytotechnologist

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What are Electron Microscopes?

• Electron Microscopes are scientific instruments
  that use a beam of highly energetic electrons to
  examine objects on a very fine scale. It has much
  higher magnification and resolving power than a
  light microscope, with magnifications up to two
  million times, allowing it to see smaller objects
  and greater detail in these objects. It is
  similar to a light microscope in using a beam of
  electromagnetic radiation condensed, focused, and
  magnified by lenses to image a specimen.
• However the EM uses much higher energy, shorter
  wavelength electromagnetic radiation compared to
  a light microscope, and the electron microscope
  uses electrostatic and electromagnetic lenses to
  control the illuminating and imaging of the
  specimen, rather than the glass lenses used by a
  light microscope.

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Where did Electron Microscopes Come From?

• Electron Microscopes were developed due to the
  limitations of Light Microscopes which are
  limited by the physics of light to 500x or 1000x
  magnification and a resolution of 0.2
  micrometers. In the early 1930's this theoretical
  limit had been reached and there was a scientific
  desire to see the fine details of the interior
  structures of organic cells (nucleus,
  mitochondria...etc.). This required 10,000x plus
  magnification which was just not possible using
  Light Microscopes.The Transmission Electron
  Microscope (TEM) was the first type of Electron
  Microscope to be developed and is patterned
  exactly on the Light Transmission Microscope
  except that a focused beam of electrons is used
  instead of light to "see through" the specimen.
  It was developed by Max Knoll and Ernst Ruska in
  Germany in 1931.The first Scanning Electron
  Microscope (SEM) debuted in 1942 with the first
  commercial instruments around 1965. Its late
  development was due to the electronics involved
  in "scanning" the beam of electrons across the
  sample.

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How do Electron Microscopes Work?

• Electron Microscopes (EMs) function exactly as
  their optical counterparts except that they use a
  focused beam of electrons instead of light to
  "image" the specimen and gain information as to
  its structure and composition.
• The basic steps involved in all EMs
• A stream of electrons is formed (by the Electron
  source) and accelerated toward the specimen using
  a positive electrical potential
• This stream is confined and focused using metal
  apertures and magnetic lenses into a thin,
  focused, monochromatic beam.
• This beam is focused onto the sample using a
  magnetic lens
• Interactions occur inside the irradiated sample,
  affecting the electron beam
• These interactions and effects are detected and
  transformed into an imageThe above steps are
  carried out in all EMs regardless of type.

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Transmission Electron Microscope (TEM)

• Yields information such as
• Morphology
• The size, shape and arrangement of the particles
  which make up the specimen as well as their
  relationship to each other on the scale of atomic
  diameters.
• Crystallographic Information
• The arrangement of atoms in the specimen and
  their degree of order, detection of atomic-scale
  defects in areas a few nanometers in diameter
• Compositional Information (if so equipped)
• The elements and compounds the sample is
  composed of and their relative ratios, in areas a
  few nanometers in diameter

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How does TEM work?

• A TEM works much like a slide projector except
  that they shine a beam of electrons (like the
  light) through the specimen(like the slide).
  Whatever part is transmitted is projected onto a
  phosphor screen for the user to see.
• The "Virtual Source" at the top represents the
  electron gun, producing a stream of monochromatic
  electrons.
• This stream is focused to a small, thin,
  coherent beam by the use of condenser lenses 1
  and 2. The first largely determines the "spot
  size" the general size range of the final spot
  that strikes the sample. The second lens
  actually changes the size of the spot on the
  sample changing it from a wide dispersed spot to
  a pinpoint beam.

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• The beam is restricted by the condenser aperture
  knocking out high angle electrons. The beam
  strikes the specimen and part of it is
  transmitted. This transmitted portion is focused
  by the objective lens into an image
• Optional Objective and Selected Area metal
  apertures can restrict the beam the Objective
  aperture enhancing contrast by blocking out
  high-angle diffracted electrons, the Selected
  Area aperture enabling the user to examine the
  periodic diffraction of electrons by ordered
  arrangements of atoms in the sample
• The image is passed down the column through the
  intermediate and projector lenses, being enlarged
  all the way and strikes the phosphor image screen
  and light is generated, allowing the user to see
  the image.
• The darker areas of the image represent those
  areas of the sample that fewer electrons were
  transmitted through (they are thicker or denser).
  The lighter areas of the image represent those
  areas of the sample that more electrons were
  transmitted through (they are thinner or less
  dense)

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• SEMs are patterned after Reflecting Light
  Microscopes and yield similar information
• Topography
• The surface features of an object or "how it
  looks", its texture detectable features limited
  to a few manometers
• Morphology
• The shape, size and arrangement of the particles
  making up the object that are lying on the
  surface of the sample or have been exposed by
  grinding or chemical etching detectable features
  limited to a few manometers
• Composition
• The elements and compounds the sample is
  composed of and their relative ratios, in areas
  1 micrometer in diameter
• Crystallographic Information
• The arrangement of atoms in the specimen and
  their degree of order only useful on
  single-crystal particles gt20 micrometers

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• Unlike the TEM, where electrons of the high
  voltage beam form the image of the specimen, the
  Scanning Electron microscope(SEM) produces images
  by detecting low energy secondary electrons which
  are emitted from the surface of the specimen due
  to excitation by the primary electron beam. In
  the SEM, the detectors building up an image by
  mapping the detected signals with beam position.
• Generally, the TEM resolution is about an order
  of magnitude greater than the SEM resolution,
  however, because the SEM image relies on surface
  processes rather than transmission it is able to
  image bulk samples and has a much greater depth
  of view, and so can produce images that are a
  good representation of the 3D structure of the
  sample

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How a typical SEM functions?

• The "Virtual Source" at the top represents the
  electron gun, producing a stream of monochromatic
  electrons.
• The stream is condensed by the first condenser
  lens. This lens is used to both form the beam and
  limit the amount of current in the beam.
• The beam is then constricted by the condenser
  aperture eliminating some high-angle electrons
• The second condenser lens forms the electrons
  into a thin, tight, coherent beam and is usually
  controlled by the "fine probe current knob"
• A user selectable objective aperture further
  eliminates high-angle electrons from the beam

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• A set of coils then "scan" or "sweep" the beam in
  a grid fashion, dwelling on points for a period
  of time determined by the scan speed (usually in
  the microsecond range)
• The final lens, the Objective, focuses the
  scanning beam onto the part of the specimen
  desired.
• When the beam strikes the sample (and dwells for
  a few microseconds) interactions occur inside the
  sample and are detected with various instruments
• Before the beam moves to its next dwell point
  these instruments count the number of
  interactions and display a pixel whose intensity
  is determined by this number (the more reactions
  the brighter the pixel).
• This process is repeated until the grid scan is
  finished and then repeated, the entire pattern
  can be scanned 30 times per second.

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Electron Source (Gun)

• All Electron Microscopes utilize an electron
  source of some kind with the majority using a
  Thermionic Gun as shown below

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A Thermionic Electron Gun functions in the
following manner

• A positive electrical potential is applied to the
  anode. The filament (cathode) is heated until a
  stream of electrons is produced.
• The electrons are then accelerated by the
  positive potential down the column.
• A negative electrical potential (500 V) is
  applied to the Whenelt Cap
• As the electrons move toward the anode any ones
  emitted from the filament's side are repelled by
  the Whenelt Cap toward the optic axis (horizontal
  center)
• A collection of electrons occurs in the space
  between the filament tip and Whenelt Cap. This
  collection is called a space charge .
• Those electrons at the bottom of the space charge
  (nearest to the anode) can exit the gun area
  through the small (lt1 mm) hole in the Whenelt
  Cap.
• These electrons then move down the column to be
  later used in imaging.

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• This process insures several things
• That the electrons later used for imaging will be
  emitted from a nearly perfect point source (the
  space charge)
• The electrons later used for imaging will all
  have similar energies (monochromatic)
• Only electrons nearly parallel to the optic axis
  will be allowed out of the gun area

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• Specimen interaction is what makes Electron
  Microscopy possible. The energetic electrons in
  the microscope strike the sample and various
  reactions can occur as shown below. The reactions
  noted on the top side of the diagram are utilized
  when examining thick or bulk specimens(SEM) while
  the reactions on the bottom side are those
  examined in thin or foil specimens (TEM)

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• Bulk Specimen Interactions
• Backscattered Electrons
• Formation Caused by an incident electron
  colliding with an atom in the specimen which is
  nearly normal to the incident's path. The
  incident electron is then scattered "backward"
  180 degrees.
• Utilization The production of backscattered
  electrons varies directly with the specimen's
  atomic number. This differing production rates
  causes higher atomic no. elements to appear
  brighter than lower atomic number elements. This
  interaction is utilized to differentiate parts of
  the specimen that have different average atomic
  number.

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Secondary Electrons

• Source Caused by an incident electron passing
  "near" an atom in the specimen, near enough to
  impart some of its energy to a lower energy
  electron (usually in the K-shell). This causes a
  slight energy loss and path change in the
  incident electron and the ionization of the
  electron in the specimen atom. This ionized
  electron then leaves the atom with a very small
  kinetic energy (5eV) and is then termed a
  "secondary electron". Each incident electron can
  produce several secondary electrons.

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• Utilization Production of secondary electrons
  is very topography related. Due to their low
  energy, 5eV, only secondary electrons that are
  very near the surface (lt10 nm) can exit the
  sample and be examined. Any changes in topography
  in the sample that are larger than this sampling
  depth will change the yield of secondary
  electrons due to collection efficiencies.
  Collection of these electrons is aided by using a
  "collector" in conjunction with the secondary
  electron detector. The collector is a grid or
  mesh with a 100V potential applied to it which
  is placed in front of the detector, attracting
  the negatively charged secondary electrons to it
  which then pass through the grid-holes and into
  the detector to be counted.

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Sample Preparation

• The technique required varies depending on the
  specimen and the analysis required
• 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. An
  entire field called cryo-electron microscopy has
  branched from this technique. With the
  development of cryo-electron microscopy of
  vitreous sections(CEMOVIS), it is now possible to
  observe virtually any biological specimen close
  to its native state.
• Dehydration - replacing water with organic
  solvents such as ethanol or acetone.
• Embedding - infiltration of the tissue with a
  resin such as araldite or epoxy for sectioning.
  After this embedding process begins, the specimen
  must be polished to a mirror-like finish using
  ultra-fine abrasives. The polishing process must
  be done accordingly, or it may lead to scratches
  imposing on the image quality.
• Sectioning - produces thin slices of specimen,
  semitransparent to electrons. These can be cut on
  an ultra-microtome with a diamond knife to
  produce very thin slices. Glass knives are also
  used because they can be made in the lab and are
  much cheaper.

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Sample Preparation

• Staining - uses heavy metals such as lead,
  uranium or tungsten to scatter imaging electrons
  and thus give contrast between different
  structures, since many (especially biological)
  materials are nearly "transparent" to electrons.
  In biology, specimens are usually stained "en
  bloc" before embedding and also later stained
  directly after sectioning by brief exposure to
  aqueous (or alcoholic) solutions of the heavy
  metal stains.

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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 (cryo-fixed), then
  fractured by simply breaking or by using a
  microtome while maintained at liquid nitrogen
  temperature.
• The cold fractured surface (sometimes "etched" by
  increasing the temperature to about -100C 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.
  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. The still-floating replica is
  thoroughly washed from residual chemicals,
  carefully fished up on EM grids, dried then
  viewed in the TEM.

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• Ion Beam Milling - thins samples until they are
  transparent to electrons by firing ions
  (typically argon) at the surface from an angle
  and sputtering material from the surface. 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.
• 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.
  Such coatings include gold, gold/palladium,
  platinum, tungsten, graphite etc. and are
  especially important for the study of specimens
  with the scanning electron microscope

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Disadvantages

• Electron microscopes are expensive to buy and
  maintain.
• They are dynamic rather than static in their
  operation requiring extremely stable
  high-voltage supplies, extremely stable currents
  to each electromagnetic coil/lens,
  continuously-pumped high-/ultra-high-vacuum
  systems, and a cooling water supply circulation
  through the lenses and pumps.
• As they are very sensitive to vibration and
  external magnetic fields, microscopes aimed at
  achieving high resolutions must be housed in
  buildings (sometimes underground) with special
  services. Newer generations of TEM operating at
  lower voltages (around 5 kV) do not have
  stringent voltage supply, lens coil current,
  cooling water or vibration isolation requirements
  and as such are much less expensive to buy and
  far easier to install and maintain.
• The samples have to be viewed in vacuum, as the
  molecules that make up air would scatter the
  electrons. Recent advances have allowed hydrated
  samples to be imaged using an environmental
  scanning electron microscope.

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• Scanning electron microscopes usually image
  conductive or semi-conductive materials best.
  Non-conductive materials can be imaged by an
  environmental scanning electron microscope. A
  common preparation technique is to coat the
  sample with a several-nanometer layer of
  conductive material, such as gold, from a
  sputtering machine however this process has the
  potential to disturb delicate samples.
• The samples have to be prepared in many ways to
  give proper detail, which may result in artifacts
  purely as the result of treatment. This gives the
  problem of distinguishing artifacts from
  material, particularly in biological samples.
  Scientists believe that electron microscopy
  features correlate with living cells.

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Differences between LM and EM
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Namaste