electron microscopy: introduction & history

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



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.).



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.



Solenoid (electromagnetic

fields that can be varied)Subatomic particles

(electrons, protons, positrons)

Concave mirror or Fresnel LensHeat



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.



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’.



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



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.



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).



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.



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”



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}



1939 - First EM built in North America by James Hillier and Albert Prebus

at the University of Toronto

Dr. Prebus Dr. Ladd



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.



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.



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“.



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



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



Some other variants:

• High Voltage Electron Microscopy {HVEM}

• High Resolution TEM {HRTEM}

• Scanning Confocal Electron Microscopy {SCEM}



Light vs Electron

Microscope

Light vs Electron

Microscope



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electron microscopy: introduction & history

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