8/3/2019 Electron Microscopy: Introduction & History
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Bhaskar Ganguly
Ph.D. Scholar (Vety. Biochemistry)
Animal Biotechnology Center
Deptt. Of Vety. Physiol. & Biochem.
C.V.A.Sc. Pantnagar. INDIA
8/3/2019 Electron Microscopy: Introduction & History
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Definitions:
Microscope - A device with a lens or series of
lenses that enlarge (magnify) the appearanceof an object.(Does not apply to SEM)
Image - Perception of an object using our eyes(vision); requires visible light. We can sense an
object without vision (touch, etc.).
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Curved glass or mirror for
Visible light
concave convex
Concave surface of metal (e.g. satellite dish)
Radio waves
Lens - A lens is an optical component which is used to focus beams of
radiation. Lenses for light are usually made of a transparent material,
whereas non-uniform electromagnetic fields are used as lens for
electrons.
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Solenoid (electromagnetic
fields that can be varied)Subatomic particles
(electrons, protons, positrons)
Concave mirror or Fresnel LensHeat
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Resolution - The point at which two or more objects can be
distinguished as separate individual objects.
Magnification - The ratio between image size to the object size; can
be varied by changing the distance between the object and the final
lens (of the eye) or by inserting a second lens between the two.
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History:
First record of using glass lens for magnification was by Al Hazen, a
Persian scientist, around 10th to 11th
century A.D. He performed the bulk of
his studies and work in Spain.
He contradicted Ptolemy's and Euclid's theory of vision that objects are seen by
rays of light emanating from the eyes.
According to Al Hazen, the rays originate
from the object of vision and not in the
eye. Because of his extensive researchon vision, he has been considered by
many as the ‘FATHER OF MODERN
OPTICS’.
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Antonie van Leeuwenhoek (dry
goods merchant) performed studies
with glass magnifiers out of curiosity, and described three shapes
of ‘Animalcules’ using his single
lens microscope (glass bead in metal
holder).
Antonie van Leeuwenhoek
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Robert Hooke (1635 – 1703):
In 1665, Hooke described cork and other
microorganisms in a drop of water.
First to produce a book on microscopicobservations.
Made several modifications creating a
compound microscope.
Few improvements were made to the light
microscope until the 19th century.
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By mid-19th century, it became evident that theoretical resolution
limits of light were reached.
Above a magnification of 1,500 resolution is lost.
The image can be magnified, but blurred (empty magnification).
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Wavelength - the distance
between peaks of the
waveform
In 1870, Ernest Abbe derived mathematical expression
for resolution of a microscope.
Resolution is limited to approx. 1/2 thewavelength of illuminating source.
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Shortest visible wavelength - Blue light has a wavelength of 0.47
um.
Resolution max = 0.2 um (200 nm)Cannot go beyond this even with better optics
Solution: Use illumination of shorter wavelength
Köhler and Rohr used UV illumination to achieve an increase in
resolving power of about a factor of two; Required more expensive
quartz optical components, due to the absorption of UV by glass.
The Big Question: ‘X-Rays’ or ‘Electron Beams’ ?
Antone de Broglie (1924): Theory of wave nature of electrons
Hermann Busch (1924): Axial magnetic fields refract electrons
“Electron optics”
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1935 - Max Knoll demonstrates the theory of the scanning electron
microscope
1939 - Ruska and von Borries, working for Siemens produce the first
commercially available EM
1938 - First scanning electron microscope
produced by Manfred von Ardenne
Knoll and Ruska
In 1931, the German physicist Ernst Ruska and
German electrical engineer Max Knoll
constructed the prototype electron microscope,
capable of four-hundred-power magnification.
{Nobel Prize in 1986}
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1939 - First EM built in North America by James Hillier and Albert Prebus
at the University of Toronto
Dr. Prebus Dr. Ladd
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Ernst August Friedrich Ruska
(1906 – 1988):
In 1933, Ruska built an electron microscopethat exceeded the resolution attainable with
an optical (lens) microscope. Family illness
compelled the electrical engineer to devise
an electrostatic microscope, because he
wanted to make visible the poliomyelitis virus.
In 1937, Siemens financed the work of Ernst
Ruska and Bodo von Borries, and employed
Helmut Ruska (Ernst’s brother) to develop
applications for the microscope, especially
with biologic specimens.
Although contemporary electron microscopes
are capable of two million-power
magnification, as scientific instruments, they
remain based upon Ruska’s prototype.
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Transmission electron microscopy (TEM) is a microscopy technique
whereby a beam of electrons is transmitted 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 fluorescentscreen, on a layer of photographic film, or to be detected by a sensor such as
a CCD camera. The first TEM was built by Max Knoll and Ernst Ruska in
1931, with this group developing the first TEM with resolving power greater
than that of light in 1933 and the first commercial TEM in 1939.
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Scanning electron microscopy (SEM) is a technique that images a sample by
scanning it with a high-energy beam of electrons in a raster scan pattern. The
electrons interact with the atoms that make up the sample producing signals that
contain information about the sample's surface topography, composition, and other
properties such as electrical conductivity. The first SEM image was obtained by
Max Knoll, who in 1935 obtained an image of silicon steel showing electronchanneling contrast. Further work on the physical principles of the SEM and beam
specimen interactions was performed by Manfred von Ardenne in 1937, who
produced a British patent but never made a practical instrument. The SEM was
further developed by Professor Sir Charles Oatley and his postgraduate student
Gary Stewart and was first marketed in 1965 by the Cambridge ScientificInstrument Company as the "Stereoscan“.
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Scanning transmission electron microscopy (STEM) is a type of transmission
electron microscopy (TEM). As with any transmission illumination scheme, the
electrons pass through a sufficiently thin specimen. However, STEM is
distinguished from conventional transmission electron microscopes (CTEM) by
focusing the electron beam into a narrow spot which is scanned over the sample
in a raster. The first STEM was built in 1938 by Baron Manfred von Ardenne,working in Berlin for Siemens. However, the results were inferior to that of TEM
at the time, and von Ardenne only spent two years working on the problem. The
microscope was destroyed in an air raid in 1944, and von Ardenne did not return
to the field after WWII. In 1970s Albert Crewe at the University of Chicago
developed the field emission gun and added a high quality objective lens tocreate the modern STEM
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Low-voltage electron microscope (LVEM) operates at accelerating
voltages of a few kiloelectronvolts (≈ 5 keV). It allows high quality images to
be produced for samples that cannot be visualized under conventional
electron microscopes. Its electron column is inversely mounted, i.e. the
source is at the bottom of the instrument. In TEM mode, the electrons aredirected up through the sample and form a pinpoint image on a screen. Light
objectives are then used to magnify the image further to the CCD camera.
The column has internal detectors for measuring backscattered electrons for
imaging in the SEM mode. The instrument also includes a photomultiplier
used to image in the STEM mode.
Key advantages include:
• Higher contrast
• Stain not required
• Multiple modes
• Resolution
• Benchtop size
• Reduced vibration sensitivity
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Some other variants:
• High Voltage Electron Microscopy {HVEM}
• High Resolution TEM {HRTEM}
• Scanning Confocal Electron Microscopy {SCEM}
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Light vs Electron
Microscope
Light vs Electron
Microscope
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