Group 5 Project - Electron Microsopy

From CellBiology
Colourised SEM of Salmonella

Introduction

One of the most important techniques in cell biology - possibly the most important - is electron microscopy as it allows for the analysis of cells, organelles, molecules and particles that are too small to be seen with the naked eye. Using an accelerated beam of electrons which has a much smaller wavelength than light and a number of electromagnetic 'lenses', an image with a very high resolution can be obtained, permitting a more in-depth analysis of the specimen.

Electron microscopes have permitted cell biologists to see cells and cell components with much greater resolution than light microscopes, providing insight into cell structure and function that would have previously been impossible. The analysis of microstructures helps to differentiate between minute elements of the specimen being investigated and also provides information about the integrity of a structure. The electron microscope has also proven important in environmental forensic investigations as well as materials research and the semiconductor industry.

There are two major types of electron microscope - the transmission electron microscope (TEM) and the scanning electron microscope (SEM).

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. [1]
  • Received the Nobel Prize in Physics, 1906

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

  • Discovered the wave nature of electrons [2]
  • Awarded the Nobel Prize in Physics in 1929 for his work.

Hans Busch - Magnetic or electric fields act as lenses for electrons (1927) [3]

  • 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
Ernst Ruska (1906 - 1988) and the development of the electron microscope [3]
  • 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.
The first electron microscope
  • Started work with Bodo von Borries in 1932, refining the magnetic converging lens of the 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 projective 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 [4] [5]

  • 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 - 1938 [4] [5]

  • Made groundbreaking research on the physical properties of the SEM 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 - 1942 [4] [5]

  • Developed a sealed- off field emission SEM and produced a primitive 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 [5]

  • In the late 1940s, Oatley, a lecturer in engineering became interested in conducting some research in electron optics and decided to pursue the SEM to complement the works of fellow colleague - V. E. Cosslett's work on the Transmission Electron Microscope.
  • 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.
One of the First 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 continued to improve the SEM, by 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 SEM designed for commercial use was developed in the early 1960's.


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, but with some differences:

A transmission electron microscope


Light Microscope Electron Microscope
Has a light source A beam of electrons replaces the light source
Has a place for the specimen The prepared specimen sits in a vacuum chamber
Has lenses to magnify the specimen The “lenses” are hoop-like coiled electromagnets that the electron beam passes through
Displays a magnified image of the specimen[6] The image is displayed on a screen, or as a photograph [6]


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. [7]


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)[8]. [9]


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. [10]


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.[9]


Transmission Electron Microscope

A simplified student-drawn schematic drawing of a transmission electron microscope

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. [11]


A transmission electron micrograph of a human striated muscle cell showing internal structure
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. [12]


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 1-2 nanometres in size (1 million times magnification).[13] Cite error: Closing </ref> missing for <ref> tag


Scanning Electron Microscope

A simplified student-drawn schematic drawing of a scanning electron microscope

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.


A scanning electron micrograph of mammalian tracheal epitheium

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. [9]


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 between 3-20 nanometres, depending on the microscope.Cite error: Closing </ref> missing for <ref> tag The SEM also has a wide field of vision, allowing more of a specimen to be viewed at any one time than when using other imaging methods. Although in the vast majority of cases the specimen to be studied must be dead in order to complete this preparation process, there is a form of the SEM called the Environmental SEM (ESEM) that can, in some cases, be used to study live specimens. In these microscopes, the electron beam is kept in a vacuum until it reaches the specimen, which is not in a vacuum, allowing it to remain alive. An ionizable gas (e.g. water) is released into the specimen chamber to amplify the signal from scattered electrons and to prevent charge building up on the specimen. However, scattering of the electron beam occurs to a much greater extent in this technique as the electron beam is not constantly in a vacuum.[14] [9]


Comparing the Two

Transmision Electron Microscope Scanning Electron Microscope
Image produced is a section through specimen Image produced is of the surface of specimen
Electron beam passes through specimen Electron beam ‘scans’ over specimen
Specimens must be very thin Specimens can be larger
Very high resolution (1-2 nanometres, or x1,000,000) Resolution not as high as TEM (3-20 nanometres)
Specimens must be dead Specimens must be dead (with some special exceptions)
All images are black and white
Microscopes are large and expensive
A vacuum is required


Current Applications

The Electron Microscope (EM) has many applications unique to itself as it has a stronger magnification power than light microscopy. This allows a better understanding of the phenomena that occur out of the range of other viewing devices. Some examples of the current uses of electron microscopy include:

Studies of cells

Providing discoveries in:

Cell Morphology and Ultrastructure

EMs provide information about surface structures giving insight into their relationship with internal structures, as well as allowing identification of cells based on their surface morphology.[15]. The ultrastructural detail obtained by findings can be applied in cell biology for a better understanding of cellular functions and the processes associated. With the use of the TEM, Morinaga et al (2009) were able to show osseointegration does not take place at the surface of bone or the titanium implants, and thus altered understanding of ultrastructural development of lamellar bone in relation to titanium implants over time [16].

Cell-Cell Communication
Cell junctions in intestinal epithelium

With the use of electron micrographs we are able to see the arrangement of gap junctions, which facilitate cell-cell communication. The connexins can be seen to take a hexagonal shape and allow small molecules to pass through [17]. Such understanding of gap junctions is only possible by knowing their structure from use of the EM.

Phagocytosis

The EM helps to display the importance of phagocytosis in the body’s reaction to foreign particles. From the micrographs in Berry and Galle's article (1980), it is clear that the alveolar pneumocytes have phagocytosed glass and clay in an attempt to eliminate the particles from the airways [18]. With the application of the EM, we are able to gain a better understanding of human phagocytes. From EM images, hypotheses have been made about other cellular components that may be involved in the phagocytic process, for example dense filaments for transports of vacuoles [19], providing leads for further research.

Link to Journal Article: Electron Microscopy of Group A Streptococci After Phagocytosis by Human Monocytes

Distribution of Ribosomes, Parasites or Proteins in Ultrathin Sections of Cell Tissue

Using the TEM, RNA probes and cell hybridisation concurrently, we are able to display the distribution of ribosomes, nuclei, mitochondria, parasites [20] and proteins across a tissue, the sites at which genes are expressed [21], and the effects that viruses have on components [22]. This is important when locating and identifying a desired subject, gaining insight about its origin and effects, and also when verifying results from other methods, such as light microscopy, by comparing consistency of results.

Structural Information of Organelles

As electron microscopy improves, so does our structural and functional knowledge of all cells including their cellular components. Where previous researchers have made assumptions on the indefinite, current researchers can analyse based on EM findings. Often the structure of mitochondria was presented as uniform yet recent research has shown that it is highly variable and that "cristae conformation is a direct consequence of the specialized function of the respective tissue." [23]. This explains the variety of structures of the mitochondria across a range of tissue types. Structures of Golgi Apparatuses have also been shown to differ greatly in different tissues, from small stacks, to large cylindrical shapes [24]. In 1984, Barnes and Blackmore clarified perceptions of the chloroplast, confirming a sharply defined envelope which connected to the inner membranous system in places, and were able to deduce exact sizes of the organelles as well [25].

Link to Journal Article: Scanning Electron-Microscopy of Chloroplast Ultrastructure

Mechanisms of Disease

In the University of California, San Francisco scientists have viewed the ultrastructure of Glaucoma using the SEM, to gain insight into the mechanisms of ocular disease. Using the TEM, they can also view intracellular conditions and components over a range of Glaucoma specimens, both in vivo and in vitro [26].

Link for Further Research University of San Francisco - Department of Ophthalmology

Here, the electron microscope provides ultrastructural information of the human papilloma virus, helping to differentiate it from other viruses

Virus Identification

In 1947, electron microscopy was used to distinguish chickenpox from smallpox as the sizes of the cells are smaller and more sparse[27]. The detail acquired about such small particles and their structures makes it possible to differentiate a wide variety of contagious agents. One downfall of the electron microscope for detecting viruses is that the images can be ambiguous, and thus the analyst must find multiple cells of the same morphology before identifying the virus and initial photographs must be kept to verify the identification[28].

Further reading: Modern Uses of Electron Microscopy for Detection of Viruses

Cryo-electron microscopy

This procedure was developed as the existing fixation techniques lead to altered structures of some specimens due to varied levels of dehydration. The alternative fixation was freezing the hydrated specimens. This made it possible to view a fully hydrated subject without separating it from the vacuum chamber - a benefit since placing the specimen outside the vacuum greatly reduces the resolution[29] [30]. Cryo-electron microscopy is particularly effective for structures that only exist under the forces of liquid water, and thus cannot be viewed in a dehydrated form [29].

Recently, there has been a significant development in the area of cryo-microscopy. On April 30th of this year a study was published in the journal Cell reporting that researchers at University of California, Los Angeles have imaged the structure of a virus (aquereovirus) at 3.3 angstroms – a level of resolution so great researchers could, in effect, “see” atoms. This is the first published occurrence of imaging of a biological specimen at such a resolution. [31][32]

The study has shed new light on the previously little-understood method by which non-envelope viruses fuse with and infect host cells. Using the high-resolution images, the researchers determined that the aquereovirus uses a priming stage during which it sheds its protective protein coating and readies itself to use a protein termed an “insertion finger” to invade a host cell.[31]

This study is an example of the exciting potential for cryo-electron microscopy to produce extremely high-resolution images of organic specimens in their native environment. [31]

Link to Article: A Cryo-EM Structure of a Nonenveloped Virus Reveals a Priming Mechanism for Cell Entry

Link to BBC News Article: Cryo-Electron Microscopy

Environmental Forensic Microscopy Investigations

In environmental forensic investigations, the EM is applied to assess hygiene and monitor the environment providing information about contaminants that are present. Dust, lead and asbestos particles, hairs, fibers and mineral grains are analysed by the EM and may be able to be connected to causes of poor hygiene, and/or be used as evidence in criminal cases, linking suspects to crime scenes [33].

Semiconductors Analysis

TEM is highly useful in the semiconductor industry for obtaining precise measurements of devices (nanometrology) and the analysis of faults that develop when manufactured (failure analysis). The semiconductors are so small that such detail can only be acquired by the EM. The devices are evaluated, providing feedback about certain materials used, the effectiveness of the semiconductor and a failure analysis [34].

Materials and Chemical Research

The application of TEM to materials research, such as crystallography, presents feedback that allows for improvements in the composition of a substance and analysis of the behaviour of a material in a given situation [35], particularly nanosized materials.

Future Applications

Apart from further developing applications that are currently in use there are some areas where little research has been undertaken, and as such a greater focus in these areas may lead to valuable future uses.

Diagnosis of Cancer

An electron micrograph which aids in the diagnosis and understanding of a breast cancer cell, its mechanisms and distribution of binding sites

Where the use of routine techniques, such as light microscopy, may lead to an incorrect diagnosis of a difficult tumour, the additional ultrastructural detail obtained from EM would increase the likelihood of an accurate diagnosis. Cell-cell communication between malignant and normal cells could be monitored, as well as the location of cancerous cells via immunogold labeling. Although there is not much research in this area it appears to hold promise for the future, as it could prove to be more cost effective than current procedures. [36]

Rapid detection of infectious agents

The EM must remain as one of the primary tools of rapid diagnostic virology, as it can be used to identify unexpected agents quickly so that they can be treated. An example of this is quick response and identification of B. anthracis in the biological terrorist letter attack on the U.S. [28].

Environmental Scanning Electron Microscopy (ESEM)

This electron microscope does not require the specimen itself to be in a vacuum and can be operated at warmer temperatures, allowing similar conditions to the specimen's normal environment. This offers a greater insight into specimens and their functions as they would be in life[15][37].

Uses At UNSW

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 different types of analysis. They also have a variety of equipment that is used for the preparation of both organic and inorganic specimens as well as equipment that prepares 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.[38]

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

Links to Other EM Research Facilities In Australia

  • Peter Mac Facility for Cancer Research [1]
  • Australian Microscopy and Microanalysis Society (AMMS Inc.) [2]
  • Electron Microscopy News, ABC: Catalyst [3]

Glossary

Angstrom: The smallest recognised division of a chemical element; approximately the distance between the hydrogen atoms in a molecule of water

Anode: Positively-charged electrode

Chloroplast: A plastid containing chlorophyll, where photosynthesis takes place

Condenser: A lens used to concentrate light or electrons on an object

Crystallography: The science of analyzing crystalline structures and functions of materials

Electron: A negative electric charged subatomic particle

Electromagnet: A temporary magnet in which the magnetic field is produced by the flow of electric current

EM: Electron microscope

ESEM: Environmental scanning electron microscope

Glaucoma: An eye disease that damages the optic nerve and impairs vision

Glutaraldehyde: A fixative for electron microscopy

Golgi Apparatus: An organelle that is active in the modification and transport of proteins

Immunogold labeling: Gold markers used for indirect labeling of a specific site

Morphology: Shape or structure

Mitochondria: An organelle responsible for producing a cell’s energy

Nanometrology: The science of measurement at the nanoscale level

Non-envelope virus: Viruses lacking an outer envelope membrane used to fuse with and infect host cells

Nuclei: A membrane-enclosed organelle containing most of the cell's genetic material

Organelle: A physical compartment of a cell

Oscillograph: A device that records the waveforms of fluctuating voltages or currents

Osmium Tetroxide: an electron-dense stain

Osseointegration: The growth action of bone tissue, as it assimilates surgically implanted devices or prostheses

Parasite: An animal or plant that lives in or on a host

Phagocytosis: The process where a cell engulfs and digests microorganisms a solid particle, such as a microorganism

Polepiece: An item of magnetic material comprising one end of a magnet that is shaped to direct the magnetic flux distributed around it

Resolution: The number of pixels per square inch in a photo or computer-generated display

Ribosome: An organelle in cells that makes proteins from amino acids and by translating messenger RNA

SEM: Scanning electron microscope

Semiconductor: A material that has a limited capacity for conducting an electric current

TEM: Transmission electron microscope

Ultrastructure: The very fine details of a structure

Vacuum: An empty space devoid of matter

Wavelength: The distance between two points over which a wave's shape repeats

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2010 Projects

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