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 appearance of 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.).

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.

Curved glass or mirror for Visible light

concave

convex

Concave surface of metal (e.g. satellite dish) Radio waves

Concave mirror or Fresnel Lens Heat

Solenoid (electromagnetic fields that can be varied) Subatomic particles (electrons, protons, positrons)

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.

Resolution - The point at which two or more objects can be distinguished as separate individual objects.

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 research on vision, he has been considered by many as the FATHER OF MODERN OPTICS.

Antonie van Leeuwenhoek

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).

Robert Hooke (1635 1703): In 1665, Hooke described cork and other microorganisms in a drop of water. First to produce a book on microscopic observations. 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. wavelength of illuminating source. 1/2 the

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 Khler 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

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}

Knoll and Ruska 1935 - Max Knoll demonstrates the theory of the scanning electron microscope

1938 - First scanning electron produced by Manfred von Ardenne

microscope

1939 - Ruska and von Borries, working for Siemens produce the first commercially available EM

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 microscope that 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 (Ernsts 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 Ruskas 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 fluorescent screen, 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 electron channeling 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 Scientific Instrument 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 to create 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 are directed 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

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