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ELECTRON MICROSCOPY

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Title: ELECTRON MICROSCOPY


1
ELECTRON MICROSCOPY
  • Ganesh Prasad Acharya
  • Cytotechnologist

2
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.

3
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.

4
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.

5
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

6
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.

7
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8
  • 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)

9
  • 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

10
  • 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

11
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

12
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13
  • 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.

14
Electron Source (Gun)
  • All Electron Microscopes utilize an electron
    source of some kind with the majority using a
    Thermionic Gun as shown below

15
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.

16
  • 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

17
  • 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)

18
  • 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.

19
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.

20
  • 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.

21
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.

22
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.

23
  • 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.

24
  • 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

25
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.

26
  • 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.

27
Differences between LM and EM
28
Namaste
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