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ELECTRON MICROSCOPYELECTRON MICROSCOPY
Presented by
sem.mov
Siddhartha Swarup JenaRAD/10-30
Ph.D. Mol. Bio & Biotech
IntroductionIntroduction
Microscopes magnify & resolve images
‘Its not how much they magnify that is key - but how well they resolve…’
Invented in 1930s, but not used much until after WW-II.
1932, produced the world's first transmission electron microscope (TEM).
German physicist Ernst Ruska and German electrical engineer Max Knoll constructed the prototype electron microscope.
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Invention of EM
In 1932, invented by E. Ruska et al. In 1986, Ruska received the Nobel Prize.
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Contd…
The transmission electron microscope (TEM) was the first type of Electron Microscope to be developed
The first scanning electron microscope (SEM) debuted in 1938 ( Von Ardenne) 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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Imaging with a simple lens
semi-angular aperture
object plane
image plane
<<< conjugate planes of focus >>>
axis of lens
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Focal length
f
f = focal length of lens
parallel
rays of light
axis
back focal plane of lens >>>
Distance between center of lens and focal point is the focal length
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Resolution
The limit of resolution of a microscope is the smallest distance between 2 points that can be seen using a microscope
This is a measure of the clarity of the image
As Magnification increases, resolution decreases.
Resolving power is inversely proportional to the wavelength of the radiation it uses
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Resolution
dmin =0.61 n sin
n = refractive index = wavelength
d
note: resolving power independent of lens properties
: for green (500nm) light dmin = c. 0.2 µm
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The Light Microscope
Series of lenses through which ordinary white light can be focused.
Optical microscopes can not resolve 2 points closer together than about half (0.45) the wavelength of the light used (450-600nm).
The total magnification is the eyepiece magnification multiplied by the objective magnification.
The maximum magnification of a light microscope is x1500 & resolution up to 0.2 m.
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The Electron Microscope Electrons (negatively charged very small
particles) can behave as waves. The wavelength of electrons is about 0.005nm
Electrons are ‘fired’ from an electron gun at the specimen and onto a fluorescent screen or photographic plate
Electrons scatter when they pass through thin sections of a specimen
There are 2 major types of electron microscopy - transmission and scanning
Both focus an electron beam onto the specimen using electromagnets
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Comparison of Optical and Electron MicroscopesComparison of Optical and Electron Microscopes
Electron microscopes are operated in vacuum because the mean free path of electrons in air is short – this mean biological samples should not degas – they can either be dehydrated or frozen
Electron microscopes have higher resolution than optical microscopes – atomic resolution is possible.
Chemical imaging and spectroscopy – mapping π and σ bonds at 1nm resolution can be done.
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Why high vacuum ?
Mean free path of electron is very short in air
Tungsten filaments burn out in air
Columns must be kept dust free
Achieved by two fold pumping:
Rotary (mechanical) pump + Diffusion pump or + turbo pump
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In transmission EM the electrons pass through the specimen
Specimen needs to be extremely thin - 10nm to 100nm
TEM can magnify objects up to 500 000 times
TEM has made it possible to see the details of interior views and discover new organelles
Cells or tissues are killed and chemically ‘fixed’ in a complicated and harsh treatment
Transmission Electron Transmission Electron
Microscope (TEM)Microscope (TEM)
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TEM contd…
Has a resolution 1000 times better than light microscope (0.2nm)
Transmitted electrons (those that do not scatter) are used to produce image
Denser regions in specimen, scatter more electrons and appear darker
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The process
Electrons are emitted by an electron gun, commonly fitted with a tungsten filament cathode
Electric field accelerates Magnetic (and electric) field control path
of electrons Electron wavelength @ 200KeV 2x10-
12 m Resolution normally achievable @
200KeV 2 x 10-10 m 2Å
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Components of a Transmission Components of a Transmission MicroscopeMicroscope
Thermionic Gun:
Electron source.
Triode or self-biasing gun
W, LaB6, CeB6
If misaligned, low intensity & other alignments may also be out
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Electron gun
Brightness = electron current by a source with unit area and unit solid angle
Bias (Wehnelt)Cylinder
Filament (20-100 KV)
Anode
stream of electrons originating from outer shell of filament atoms
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Lenses
Provide means to (de)focus the electron beam on the specimen, to focus the image, to change the magnification, and to switch between image and diffraction
Electromagnetic lenses are based on the fact the moving electrons are forced into a spiral trajectory, i.e. focused into one point
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The electromagnetic lens
Works at fixed focal distance and variable focal length.
(like the human eye lens but unlike light optics)
windings
soft iron pole piece
windings
e-
electrons are charged, and are therefore deflected when they cross a magnetic field
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Lens systema) Condenser lens: Uniformly illuminate the sample. Usually 2; C1 and C2 lens If misaligned, we will lose the beam when changing
magnification
b) Objective lens: Image sample – determines resolution. If misaligned, the image will be distorted, blurry.
c) Projector lens: magnifies image/ forms diffraction pattern – should not alter
resolution. If misaligned, the image will be distorted, diffraction pattern
may be blurry.S S Jena
Sample preparation
1. Chemical fixation: Proteins with formaldehyde and glutaraldehyde and
lipids with osmium tetroxide.
2. Cryofixation: Freezing a specimen so rapidly, to liquid nitrogen or even
liquid helium temperatures, that the water forms vitreous (non-crystalline) ice.
3. 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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Contd…4. Embedding The tissue is passed through a 'transition solvent' such as
epoxy propane and then infiltrated with a resin such as Araldite epoxy resin
Tissues may also be embedded directly in water-miscible acrylic resin
After the resin has been polymerised (hardened) the sample is thin sectioned (ultrathin sections) and stained - it is then ready for viewing.
5. Sectioning These can be cut on an ultramicrotome with a diamond
knife to produce ultrathin slices about 60-90 nm thick. Disposable glass knives are also used because they can be
made in the lab and are much cheaper. S S Jena
6. Staining Uses heavy metals such as lead,
uranium or tungsten to scatter imaging electrons and thus give contrast between different structures, since many biological materials are nearly "transparent" to electrons (weak phase objects).
Contd…
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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 (cryofixed), then fractured by simply breaking or by using a microtome while maintained at liquid nitrogen temperature.
The cold fractured surface 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.
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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 fine grids, dried then viewed in the TEM.
Contd…
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TEM images
Transmission electron micrograph of epithelial cells
from a rat small intestine. Scale bar = 5 mm.
TEM view of a plant cell
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TEM Limitations
Specimen dead. Specimen preparation uses
extreme chemicals so artifacts are always a concern.
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Live specimens possible. No sectioning is required.
Magnify objects up to two million times.
Lower magnifications than the TEM.
Resolving power is about 20nm
In Scanning EM microscopes the electrons bounce off the surface of the specimen
Produce images with a three-dimensional appearance
Allow detailed study of surfaces.
Scanning Electron Microscope Scanning Electron Microscope
(SEM)(SEM)
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Sample preparation
All water must be removed from the samples because the water would vaporize in the vacuum.
All metals are conductive and require no preparation before being used.
All non-metals need to be made conductive by covering the sample with a thin layer of conductive material.
This is done by using a device called a "sputter coater.“
E.g. gold coating
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EM VariationsEM Variations
High Voltage TEM Scanning tunneling microscope Scanning transmission electron
microscope (STEM) Scanning probe microscope Atomic force microscope Environmental scanning electron
microscope Elemental Composition SEM
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ApplicationsApplications
Morphology (imaging) Crystal structures (diffraction) Protein localization Electron & Cellular tomography Toxicology Biological production and viral load monitoring Particle analysis Materials qualification Structural biology Virology Forensics Mining (mineral liberation analysis)
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Disadvantages of EMDisadvantages of EM
Expensive to build and maintain
Requires extremely stable high-voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high- or 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, must be housed in stable buildings (sometimes underground) with special services such as magnetic field cancelling systems.
The samples largely have to be viewed in vacuum
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Light Vs Electron Microscopes
Feature Light Microscope Electron Microscope
Radiation used
Radiation source
Nature of lenses
Lenses used
Image seen
Radiation medium
Magnification
Limit of resolution
What it can show
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