Group 5 Project - Electron Microsopy

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Introduction

One of the most important techniques in cell biology – possibly the most important – is microscopy, since the details of cells and their organelles are too small to be seen with the naked eye. Electron microscopes have permitted cell biologists to see cells and cell components with much greater resolution than light microscopes, allowing a much greater understanding of cell structure and function.


How It Works - The Electron Microscope

There are two main types of electron microscope: the Transmission Electron Microscope (TEM) and the Scanning Electron Microscope (SEM), both of which are covered here. Before looking at each individually, a general overview of electron microscopes will be given.


Looking at an electron microscope for the first time, understanding how it works can seem like a daunting task. However, it is easier to grasp the basic concept once one realises that the electron microscope follows the same basic pattern as the light microscope. Consider a typical light microscope. A light microscope:

A transmission electron microscope


1. Has a light source

2. Has a place for the specimen

3. Has lenses to magnify the specimen

4. Displays a magnified image of the specimen[1]


In general, electron microscopes follow the same pattern, but with some differences: a

1. A beam of electrons replaces the light source

2. The prepared specimen sits in a vacuum chamber

3. The “lenses” are hoop-like coiled electromagnets that the electron beam passes through

4. The image is displayed on a screen, or as a photograph [2]


A beam of electrons fired from an electron gun is used because a fast-moving electron beam has a much smaller wavelength than light, allowing the electron microscope to have a much higher resolution than the light microscope. Commonly, a tungsten filament is used in the electron gun; a very high voltage (ranging from 50,000 to millions of volts) is passed through the filament, causing electrons to be thrown off. An anode (a positively-charged electrode) is used to accelerate the electron beam, which reduces the wavelength and allows for higher resolution images. [3]


Preparation of specimens is different for transmission and scanning electron microscopy. However, in both transmission and scanning electron microscopy, the electron beam and the specimen must be kept in a vacuum, since electrons can lose their energy to particles in air. Thus, neither TEMs nor SEMs can be used to observe live specimens, as living creatures could not survive the required fixation/preparation process, or being positioned in a vacuum (though there are some exceptions to this rule in the case of SEMs)[4]. [5]


In a light microscope, the lenses concentrate the light into a beam and magnify the image of the specimen by refracting that beam of light. The electromagnetic “lenses” of the electron microscope (coils of wire through which a current of one amp or less is passed) act in a similar way, whereby the magnetic field generated by the lenses bends and focuses the electron beam passing through it. [6]


Finally, the electrons containing information about the specimen are interpreted into an image that can be viewed either as a photograph image or on a cathode-ray TV screen. The way images are formed differs between TEMs and SEMs. [7]


Transmission Electron Microscope

(DIAGRAM) TEMs give us the ability to study the internal structure of cells and organelles. Specimens being prepared for TEMs must be fixed and sliced into very thin sections. Fixation with glutaraldehyde followed by osmium tetroxide (which acts as a fixative and an electron-dense stain) is common in electron microscopy, though there are a range of fixation techniques available, such as fixation with formaldehyde or cryo-fixation – the method of fixation depends on the specimen to be viewed and the view desired. Specimens for electron microscopy must be stained with an electron-dense substance (like lead, osmium or gold) because otherwise the accelerated electron beam can damage the specimen. [8]


Electrons are fired from the electron gun into a vacuum, where the condenser lenses focus the beam of accelerated electrons on the specimen. The electrons interact with and pass through the specimen to reach the objective lens, which produces the first image of the specimen and performs the first focusing and magnification. Next is the intermediate lens which controls magnification of the image, followed by the projector lenses, which project the final image onto an imaging device such as a photographic plate, fluorescent screen or CCD camera. The image is viewed through a leaded window or magnifying binocular eyepieces, or the image is captured by camera and viewed on an external screen. [9]


Areas of the image appearing as bright are less dense, so more electrons were able to pass through, while dark areas indicate dense areas of the specimen where fewer electrons were able to pass through. Images produced by TEMs are two dimensional. TEMs have the highest resolution of any electron microscope, allowing us to see structures 1 nanometre in size (1 million times magnification)[10].[11]


Scanning Electron Microscope

(DIAGRAM) Since SEMs give us an image of the surface of a specimen, preparation of specimens for scanning electron microscopy differs from that of transmission electron microscopy. Larger specimens can be placed in an SEM than can be placed in a TEM (e.g. whole insects) as the beam does not have to pass through the specimen but over it. Specimens must be coated with a metal that reflects electrons, such as gold; this coating of a conductive substance amplifies the electron signal and also prevents the electrons from charging the specimen. [12]

Electrons are fired from the electron gun into the vacuum and accelerated before they pass through one or two condenser lenses that focus the electron beam. The beam then passes through magnetic coils controlled by a scanning generator that direct it in a predetermined scanning motion over the surface of the specimen. As electrons hit the surface of the specimen they bounce off – these are known as secondary electrons. The secondary electrons are detected by an electron collector which is connected to an amplifier, which processes and amplifies the signal before it is sent to a cathode-ray tube screen where the image can be viewed. The scanning generator also scans the electron beam in the cathode ray tube, and allows magnification of the view displayed on the cathode-ray tube screen by reducing the surface area of the specimen being scanned.[13]

SEMs produce very sharp three-dimensional images of the surfaces of specimens; however, the resolution is less than that of TEMs, with maximum resolution being approximately 10 nanometres[14].[15]


Comparing the Two

Transmision Electron Microscope Scanning Electron Microscope
Electron beam passes through specimen Electron beam ‘scans’ over specimen
Specimens must be very thin Specimens can be larger
Very high resolution (1 nanometre, or x1,000,000) Resolution not as high as TEM (about 10 nanometres)
Specimens must be dead Specimens must be dead (but some special exceptions)
All images are black and white
Microscopes are large and expensive
A vacuum is required


History

J.J Thompson (1856 - 1940) - Discovery of the Electron

  • In 1897 during a series of experiments that were set up to study the nature of electric discharge in a high-vacuum cathode ray tube, he discovered the electron. [16]
  • Received the Nobel Prize in Physics, 1906

Louis deBroglie (1892 - 1987) - Wavelength of moving electrons

  • Discovered the wave nature of electrons [17]
  • Awarded the Nobel Prize in Physics in 1929 for his work.

Hans Busch - Magnetic or electric fields act as lenses for electrons [18]

  • Calculated the electron trajectories in an electron ray bundle and found that a magnetic field of the short coil had the same effect on the electron bundle as a convex glass lens has on a light bundle.

Ernst Ruska (1906 - 1988) and the development of the electron microscope [19]

Ernst Ruska
  • In 1929 he submitted his Student Project Thesis on a method of designing a cathode ray oscillograph on the basis of the experimentally found dependence of the writing spot diameter on the position of the concentrating coil.
  • In 1930 he published a paper on the contribution to geometrical electron optics.
  • In 1931 the first blueprints of the electron microscope were put together by Ernst Ruska and Max Knoll
  • Ernst Ruska and Max Knoll published Das Elektronenmikroskop (The electron microscope) in 1932, announcing the development of the electron microscope to the scientific world.
  • started work with Bodo von Borries in 1932, refining the magnetic converging lens of short field lens needed to obtain a better than light resolution.
  • In 1937 along with Bodo von Borries, began collaborating with Siemens to industrially produce the electron microscope.
  • By 1938 two prototypes with a condenser and polepieces for objective and projectinve as well as airlocks for specimens and photoplates had been completed, with a maximum resolution of 30,000x
  • In 1939 the first serially produced electron microscope was delivered to IG Farbenindustrie, a major representative of the chemical industry, at its works in Hoeschst.
  • In 1986 Ernst Ruska was awarded the Nobel Prize in Physics for his work in electron optics and the design of the first electron microscope.

M. Knoll - First Scanning Electron Micrograph [20] [21]

  • In 1935, Knoll used a primitive version of a SEM which contained two cathode ray tubes and produced a micrograph of a solid polycrystalline structure which was a piece of steel.

Manfred von Ardenne (von Ardenne, 1938)

  • Made ground-breaking research on the physical properties of the Scanning Electron Microscope and beam specimen interactions.
  • He developed a British patent SEM but it was never made into a working model.

Zworykin et al. - first early form of SE Image [22] [23]

  • Developed a sealed- off field emission SEM and produced an primative SEM image. However it did not meet up with the swiftly developing Transmission Electron Microscope and so was considered uneventful and further progress was terminated.

Professor Sir Charles Oatley (1904-1996) and his involvement in the development of the Scanning Electron Microscope [24]

  • In the late 1940s, Oatley, a lecturer in engineering became interested in conducting some research in electron optics and decided to persue the SEM to complement the works of fellow colleague - V. E. Cosslett's work on the TEM.
  • In 1948, with one of his students - Dennis McMullan, built their first SEM. With this microscope they could achieve a resolution of 50nm, and were able to produce the first micrographs showing the striking 3D imaging characteristics of the modern-day SEM
  • In 1952, Oatley began work with another student, Ken Smith. Together they continued to make improvements to the electron optical system and increase the efficiency of secondary electron collection. In 1955 they published 'The Scanning Electron Microscope its Fields of Application', which outlined the uses of the SEM.
  • A third research student, O. C. Wells started work with Oatley in 1953. He developed the second SEM which had many improvements on the original SEM. These improvements made the SEM better for experimental work and the configuration was used in all subsequent SEMs.
  • His fourth student, Everhart who started in 1955, worked with Oatley and together they worked on tweaking the SEM to make it more efficient and improve the quality of the images produced.
  • With his fifth student Peter Spreadbury in 1956, they built a simple SEM which utilised CRT as a display unit.
  • Between 1956 and 1960, Oatley supervised a number of other students who further improved the SEM, such as applying ion beam optics, adding a magnetic objective lens which improved resolution, modifying the SEM built by Wells to enable the examination of thermionic emitters at temperatures exceeding 1000K and achieving a resolution of 10nm.
  • The first commercial SEM was developed in the early 1960's.

Current and Future Uses

The electron microscope has many applications unique to itself as it has a stronger magnification power than light microscopy. This allows a better understanding of the phenomenon that occur out of the range of other viewing devices.

Current Applications

Uses of EM include:

Studies of cells in vivo and in vitro providing discoveries in

  1. cell morphology
  2. ultrastuctural information
  3. cell to cell communication
  4. phagocytosis
  5. surface information
  6. distribution of ribosomes, parasites or proteins in ultrathin sections of cell tissue
  7. strucural information of organelles

Virus Identification

Future Applications

====Uses At UNSW==== [25]

The University of New South Wales has its' own Electron Microscope Unit (UNSW Analytical Centre, 2010). It is located in the Basement of the Chemical Science Building on the main campus.

In The EMU, there are two TEMs and five SEMs available to use, all of which are used for a different type of analysis. They also have a variety of equipment that are used for the preparation of both organic and inorganic specimens as well as equipment that prepare specimens for use in SEM and TEM examinations.

The EMU is used for research services, training and programs. It provides microscopy services to researchers from the UNSW community as well as other universities and other research bodies. The EMU is used in the collection of data for over 300 peer-reviewed papers per year. This unit educates researchers and academics through seminars, training courses and workshops as well as hands on practical experience.

To access the EMU at UNSW web site click on the link: EMU at UNSW

References

  1. Woodford, Chris. (2009) How Electron Microscopes Work Explain That Stuff. Accessed 21/4/2010 <http://www.explainthatstuff.com/electronmicroscopes.html>
  2. Woodford, Chris. (2009) How Electron Microscopes Work Explain That Stuff. Accessed 21/4/2010 <http://www.explainthatstuff.com/electronmicroscopes.html>
  3. Hunter, Elaine. (1993) Practical Electron Microscopy: A Beginner's Illustrated Guide (2nd ed.) New York and Oakleigh: Cambridge University Press. pp 114-115. isbn= 0-521-38539-3
  4. Hunter, Elaine. (1993) Practical Electron Microscopy: A Beginner's Illustrated Guide (2nd ed.) New York and Oakleigh: Cambridge University Press. p 107. isbn= 0-521-38539-3
  5. Nixon, W.C (1971). The General Principles of Scanning Electron Microscopy in Huxley, H.E and Klug, A. New Developments in Electron Microscopy. London: The Royal Society. pp. 45-50.
  6. Weakly, Brenda S. (1972) A Beginner's Handbook in Biological Electron Microscopy Edinburgh: Churchill Livingstone. pp. 4-7. ISBN 0443009082
  7. Nixon, W.C (1971). The General Principles of Scanning Electron Microscopy in Huxley, H.E and Klug, A. New Developments in Electron Microscopy. London: The Royal Society. pp. 45-50.
  8. Hunter, Elaine. (1993) Practical Electron Microscopy: A Beginner's Illustrated Guide (2nd ed.) New York and Oakleigh: Cambridge University Press. pp 3-7. isbn= 0-521-38539-3
  9. Hunter, Elaine. (1993) Practical Electron Microscopy: A Beginner's Illustrated Guide (2nd ed.) New York and Oakleigh: Cambridge University Press. pp 103-107, 114-112. isbn= 0-521-38539-3
  10. Woodford, Chris. (2009) How Electron Microscopes Work Explain That Stuff. Accessed 21/4/2010 <http://www.explainthatstuff.com/electronmicroscopes.html>
  11. Hunter, Elaine. (1993) Practical Electron Microscopy: A Beginner's Illustrated Guide (2nd ed.) New York and Oakleigh: Cambridge University Press. pp 122-130. isbn= 0-521-38539-3
  12. Nixon, W.C (1971). The General Principles of Scanning Electron Microscopy in Huxley, H.E and Klug, A. New Developments in Electron Microscopy. London: The Royal Society. pp. 45-50.
  13. Nixon, W.C (1971). The General Principles of Scanning Electron Microscopy in Huxley, H.E and Klug, A. New Developments in Electron Microscopy. London: The Royal Society. pp. 45-50.
  14. Woodford, Chris. (2009) How Electron Microscopes Work Explain That Stuff. Accessed 21/4/2010 <http://www.explainthatstuff.com/electronmicroscopes.html>
  15. Nixon, W.C (1971). The General Principles of Scanning Electron Microscopy in Huxley, H.E and Klug, A. New Developments in Electron Microscopy. London: The Royal Society. pp. 45-50.
  16. Harre R. Great Scientific Experiments: Twenty Experiments that Changed our View of the World 2002,p171, Dover Publications, USA, accessed on the 19/04/2010, [1]
  17. de Broglie, L ,1929, The Wave Nature of the Electron: Nobel Lecture, December 12, 1929, Nobel Lectures, Physics 1922-1941, Elsevier Publishing Company, Amsterdam, 1965 Accessed at | Nobelprize.org
  18. Ruska, E 1987, The Development of the Electron Microscope and of Electron Microscopy, Reviews of Modern Physics, Vol 59 Issue 3 pp 627-638
  19. Ruska, E 1987, The Development of the Electron Microscope and of Electron Microscopy, Reviews of Modern Physics, Vol 59 Issue 3 pp 627-638
  20. Wells OC. and Joy DC.The early history and future of the SEM, Surface and Interface Analysis 200b, Vol38 Issue 12-13 pp 1738-1742,
  21. Breton, B 2009, The Early History and Development of The Scanning Electron Microscope, Scientific Imaging Group at Cambridge University Engineering Department, Accessed 18/04/2010, [2]
  22. Wells OC. and Joy DC.The early history and future of the SEM, Surface and Interface Analysis 200b, Vol38 Issue 12-13 pp 1738-1742,
  23. Breton, B 2009, The Early History and Development of The Scanning Electron Microscope, Scientific Imaging Group at Cambridge University Engineering Department, Accessed 18/04/2010, [3]
  24. Breton, B 2009, The Early History and Development of The Scanning Electron Microscope, Scientific Imaging Group at Cambridge University Engineering Department, Accessed 18/04/2010, [4]
  25. UNSW Analytical Centre 2010, Homepage of Electron Microscope Unit UNSW Analytical Centre, UNSW. Accessed 22/4/2010 [5]

Breton, B 2009, The Early History and Development of The Scanning Electron Microscope, Scientific Imaging Group at Cambridge University Engineering Department, Accessed 18/04/2010, <http://www2.eng.cam.ac.uk/~bcb/history.htm>

Ruska, E 1987, The Development of the Electron Microscope and of Electron Microscopy, Reviews of Modern Physics, Vol 59 Issue 3 pp 627-638

de Broglie, L ,1929, The Wave Nature of the Electron: Nobel Lecture, December 12, 1929, Nobel Lectures, Physics 1922-1941, Elsevier Publishing Company, Amsterdam, 1965 Accessed at | Nobelprize.org

Harre, R 2002, Great Scientific Experiments: Twenty Experiments that Changed our View of the World, p171, Dover Publications, USA, accessed on the 19/04/2010, <http://books.google.com.au/books?id=yTqoV1aJWtkC&pg=RA1-PA171&dq=jj+thomson+discovery+of+the+electron&client=firefox-a&cd=2#v=onepage&q=jj%20thomson%20discovery%20of%20the%20electron&f=false>

UNSW Analytical Centre 2010, Homepage of Electron Microscope Unit UNSW Analytical Centre, UNSW. Accessed 22/4/2010 <http://srv.emunit.unsw.edu.au/Index.htm>

2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer