ELECTRON MICROSCOPY FOR BIOLOGISTS

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ELECTRON MICROSCOPY FOR BIOLOGISTS

• INTRODUCTION
• Schematiska bilder är mestadels från
  www.matter.org.uk/tem

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What is microscopy?

• Microscopy techniques - produce images at greater
  than life size -
• Magnification, M
• M D/d (size of feature in the image and d the
  size of feature in object).

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The purpose of microscopy is insight, not
images - R.W. Hamming

• Understanding of biological systems is derived
  from a knowledge of the spatial and temporal
  arrangement of structural entities ranging in
  size from organisms to macromolecules.

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The scale of things
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Proportions
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Detection

• light microscopy photons interact with the
  specimen
• electron microscopy electrons interact with the
  specimen TEM and SEM
• scanning probe microscopy utilizes a variety of
  different interactions of a fine tip with the
  specimen. Atomic forces

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Resolving power line
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Visualization

• Obtaining an image.
• Record the images for analysis or for
  documentation purposes.
• Film recording is the only technique available on
  older equipment.
• Silver based photographic film, expensive,
  enables good resolution.
• Modern instruments use digital image capture.
• Transferred to a computer screen, stored or
  printed
• Digital images are readily accessible for
  subsequent computer analysis.

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Analysis

• Counting or measuring of objects in the images.
• May involve considerable computer processing of
  the images before the objects can be analysed.
• 3-D modelling

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Physical limitations

• In transmission electron microscopy electrons
  pass through thin specimens (50-1000 nm). BULK
  BEAM
• In scanning electron microscopy signals emitted
  from the surface of thick specimens. NARROW BEAM

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why do we bother with clumsy electron-microscopy?

• The resolution of a microscope smallest
  distance between two points in the specimen gt
  perceived as separate in the image.
• resolution is determined by the wavelength of the
  radiation used.
• resolving power of a particular system (Rayleigh
  criterion)
• DR 0.61 l / N.A.
• where DR is the resolution
• l is the wavelength of the radiation used
• Numerical Aperture sin of half the angle of
  the cone of radiation entering the lens
• Resolution gain by a shorter wavelength or
  collect more data from your specimen by having
  a larger numerical aperture.

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• RADIO WAVES MICROWAVES INFRARED VISIBLE
  ULTRAVIOL. X-RAYS g -rays

• visible wavelength 390nm 750nm.
• Many cellular structures are much smaller than
  this.
• It is impossible to make lenses with a NA gt 1.4
  
• limit of resolution due to aberrations is 170nm.
  
• high absorbance for shorter wavelength radiation
• UV lenses of quartz or fluorite are expensive

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electrons accelerated by 60 KVl 0,003 nm
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TEM column
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The way forward is to use electrons

• Short wavelength given by the de Broglie
  wavelength equation
• E hc/l
• E the kinetic energy of the particle,h
  Plancks constant, c velocity of light in a
  vacuum l the wavelenth of the particle.
• e- beams can readily be generated, using an
  electron gun.
• because of their charge they can be focused by
  electromagnetic lenses.
• Electrons can be generated The kinetic energy
  they acquire is given by E Vq
• V the accelerating voltage q the charge on
  the electron

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elektronkanon
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Elektronmikroskopi på web

• http//www.matter.org.uk/tem/
• http//nobelprize.org/physics/educational/microsc
  opes/tem/index.html

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Transmission electron microscopes comparable to
light microscopes

• Electron beam path
• electron gun generates a beam
• condensor controls illumination
• objective lens magnifies image of the object
• Projector lenses, like an eyepiece, magnifies
  further

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Constraints on the use of electrons

• Vacuum is essential Moderate energy (60-200keV)
  electrons have a path length of a few mm in air
• Electron optical systems require a path 1 meter
  
• The sample cant be volatile or wet
• The absorbance of a material for electrons
  depends on its density.
• The practical thickness limit is lt 500nm for
  200kV electrons.
• Electrons are charged and interact strongly with
  matter ionize.
• If electrons accumulate in the specimen it will
  repel the electrons in the beam. (worst in SEM)
• electrons falling on the specimen must be
  conducted to earth. C or Au sputtering
• Electron beam has considerable kinetic energy, ?
  specimen temperature. Potentially resulting in
  damages.

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Lens effect

• A strong magnetic field is generated by passing
  a current through a set of windings.
• This field acts as a convex lens, bringing off
  axis rays back to focus.
• Focal length can be altered by changing the
  strength of the current.
• The image is rotated, to a degree that depends
  on the strength of the lens.

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Magnification
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Lens magnification
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Double condenser system
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1st condenser
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2nd condenser
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Condenser aperture
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Objective aperture
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Dark field and diffraction
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Intermediate lens
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Illumination adjustments
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Total magnification
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Depth of field
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Smaller aperture
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Beam alignment
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Beam alignment
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Stigmators
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Interaction with the specimen
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Electrons interact
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The preparation of the material

• key importance that the preparation of the
  material be properly controlled.
• should be applied with understanding.

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Specimen preparation