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Let us make an in-depth study of the electron microscope. After reading this article you will learn about: 1. Principle of Electron Microscope 2. Transmission Electron Microscope (Tem) 3. Components of Electron Microscope 4. Preparation of Specimen 5. Image Viewing, Development and Recording Techniques 6. Use of Electron Microscope 7. High Voltage Modern Electron Microscope 8. Scanning Electron Microscope and 9. Phase Contrast Microscope.
Introduction to Electron Microscope:
In electron microscope, high-speed electron beam is used instead of light waves, which are used in optical microscope. Like light, the stream of electrons has a corpuscular and vibratory character. Electron microscope gives very high magnification and incredibly high resolution.
The first transmission electron microscope was developed by Ernst Ruska and Max Knoll of Germany in 1931. EM is a remarkable research tool of twentieth century. It opened up subcellular structures, which were unknown to biologists. It can magnify an object upto 1000000X (one million times). The photomicrographs can be further enlarged and studied by using modern photographic techniques and computer aided techniques.
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The electrons can be focused by electro-magnetic lenses much like the light rays. Electron beam can vibrate like light rays but has very short wave length as compared to light rays. Wave length of electron beam λ = 0.005 nm as compared to 550 nm of visible light. Resolution increases with the decrease of wave length.
Resolution is dependent upon the wavelength of radiations. Smaller the radiation, greater will be the resolution. It is inversely proportional relation. The resolution determines the level of details that can be viewed from the specimen. It provides remarkable pictures with fine details.
Light microscope can achieve a maximum resolution of about 0.2 µm or 200 nm. Whereas EM can achieve a resolution of 0.10 nm which is 2000 times better than the best resolution by light microscope. Resolution is the ability of a lens to separate or distinguish between closely positioned small objects.
Principle of Electron Microscope:
Electrons are subatomic particles, which orbit around the atomic nucleus. When atoms of a metal are excited by heat energy, electrons fly off from the atom. In electron microscope, tungston is heated by applying a high voltage current, electrons form a continuous stream, which is used like a light beam.
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The lenses used in EM are magnetic coils capable of focusing the electron beam on the specimen and illuminating it. The strength of the magnetic lens depends upon the current that flows through it. Greater the flow of the current, greater will be strength of the magnetic field. The electron beam cannot pass through the glass lens.
Transmission Electron Microscope (Tem):
It consist of a system of electromagnetic lenses mounted in a column.
Components of Electron Microscope:
EM is in the form of a tall column which is vertically mounted.
It consists of the following main components:
1. Electron gun
2. Electromagnetic lenses—three sets.
3. Image viewing and recording system.
Electron gun is a heated tungsten filament, which generates electrons. Condenser lens focuses the electron beam on the specimen. A second condenser lens forms the electrons into a thin tight beam.
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To move electrons down the column, an accelerating voltage is applied between tungsten filament and anode. Now most EMs use accelerating voltages between 100 kV-1000 kV. Electrons also function as a source of illumination for the specimen. High velocity electrons pass into the system of condenser lenses, which focus them on the specimen.
The specimen to be examined must be extremely thin, at least 200 times thinner than those used in optical microscope. Ultra thin sections of 20-100 nm are cut. The specimen holder is an extremely thin film of carbon or collodion held by a metal grid.
The electronic beam passes through the specimen and electrons are scattered depending upon the thickness or refractive index of different parts of the specimen. The denser regions in the specimen scatter more electrons and therefore appear darker in the image since fewer electrons strike that area of the screen. In contrast, transparent regions are brighter.
The electron beam coming out of the specimen passes down the second of magnetic coils called objective lens, which has high power and forms the intermediate magnified image. Finally, a third set of magnetic lenses called projector (ocular) lenses produce the final further magnified image.
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Each of these lenses acts as image magnifier all the while maintaining an incredible level of details and resolution. He whole image remains in focus. This image is projected on a fluorescent screen. Below the fluorescent screen is a camera for recording the image. These lenses provide immense magnification and resolution.
As the EM works in vacuum, the specimen should be completely dry. Air molecules present in the column of EM scatter the electrons causing flicker in the electron beam. Vacuum is created in two steps. Firstly, a mechanical vacuum pump is used to create vacuum. Secondly, a diffusion pump uses a fast downward moving liquid, either oil or mercury which traps air and gas in the column. In this way, ultra high vacuum is created.
Preparation of Specimen:
The specimen have to be specially prepared for EM studies. There are various techniques for studying the specimen under EM. Some of which are discussed here.
Fixation and Dehydration:
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The specimens are fixed in glutaraldehyde, osmium tetroxide to stabilize the cell structure. After fixation, dehydration is carried out slowly with organic solvents like acetone and ethanol.
Embedding:
Resins such as araldite and epoxy are used for this purpose. Microbes are embedded in plastic resin. The specimen is soaked in un-polymerized, liquid epoxy plastic until it is completely permeated and then is hardened to form a solid block.
Ultra sectioning:
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To obtain extremely thin sections from this plastic block, Ultra-microtomes with diamond knife or glass knives are used.
Staining:
Specimens are stained with heavy metals such as lead, uranium, phosphotungstic acid. The thin sections soaked in solutions of heavy metals like lead citrate, uranyl acetate or osmium tetroxide is also used for staining.
There are some additional techniques for preparation and study of various specimens and materials.
Image Viewing, Development and Recording Techniques:
The image formed in EM is real as compared to the virtual image in optical microscope. The highly magnified image is formed below the projector lens on a fluorescent screen. Below this screen, a camera or a film or light sensitive sensor such as charge-coupled device (CCD) camera are placed. The image can be displayed on computer or monitor. For direct viewing monocular or binocular viewing, lenses are used.
The final image formed will always be in focus and needs no adjustments. The image recording and studying have undergone revolutionary changes. Digital cameras and computers have come to play a major role.
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Instead of one picture of one section, series of sections are studied and analysed. By computer aided averaging techniques of numerous images three- dimensional reconstructions of cell organelles of highest clarity are developed. Tilting of specimen also provides three-dimensional picture.
Use of Electron Microscope:
Invention of EM has come as a boon for biological sciences and industry. There is hardly any area of science that has not gained from the use of electron microscope. Immense magnification, high resolution has opened new vistas in research in cellular and molecular biology.
Study of microorganisms like bacteria, virus and other pathogens have made the treatment of diseases very effective. Fields of medicine, pathology, human anatomy have gained immensely from electron microscope studies. Health field has benefited tremendously. Nanotechnology studies are the result of electron microscope studies.
Science of microbiology owes its development to electron microscope. It also helps in tumour identification, biopsy, study of cells, variety of molecules. In industry high resolution. 2D and 3D imaging. In foresenic, mining, chemical and petrochemical industries.
High Voltage Modern Electron Microscope:
The recently developed EMs use accelerating voltages between 500-3000 kV. These EMs are used in study of metallurgy, biological materials and living cells. Thick sections upto 5µ m can also be studied.
Scanning Electron Microscope:
Transmission electron microscope (TEM) and scanning electron microscope (SEM) work on the same basic principle. TEM forms image when radiations pass and are transmitted through the specimen. Whereas SEM produces images by detecting secondary electrons which are emitted from the surface of the specimen due to excitation by the primary electron beam. Therefore SEM is used to examine the surfaces of the microorganisms in great detail.
Secondary electrons hit a scintillator which emits light which is converted into electrical current and amplified. The signal is sent to a cathode ray tube and forms image like the TV picture.
SEM was first introduced in 1965. It produces three-dimensional image, which has high resolution and great depth of field.
Electron beam passes through a pair of scanning coils or deflector plates in the electron column of the SEM in the final lens which deflects the beam on the surface of the specimen. The electrons interact with atoms on the surface of the specimen producing signals that contain information about the properties of the specimen.
The signals produced in an SEM include secondary electrons, back scattered electrons (BSE). X-rays, transmitted electrons and light. The signals scan the surface of the specimen from side to side, in lines from top to bottom. This is known as roster scanning. They produce high resolution images of the specimen surface and provide information about the composition of the specimen.
Phase Contrast Microscope:
Normally cells are studied after fixation and staining of the tissues. Only dead cells can be studied in optical microscopes. But phase contrast microscope provide a technique for studying transparent cells without staining. Different organelles appear in various shades of grey depending upon the thickness and difference between refractive index of the object and the medium. This microscope is most suitable for studying cells cultured in vitro, during mitosis, protozoans, living cells etc.
Phase contrast microscope is based upon the phase contrast principle of Fredrick Zernike who developed phase contrast microscope called Zernike microscope in 1933. He was awarded Nobel Prize in 1953 for the discovery of phase contrast principle.
If direct rays and diffracted rays of an object are in phase—having same amplitude and frequency with each other, the resultant increased amplitude is doubled. It is called constructive interference or coincidence. The object appears brighter than the surroundings.
On the other hand if direct and diffracted rays are out of phase, it is called destructive interference or interphase. The rays are partially cancelled and the object appears dim and darker than the surroundings. Both these phenomena are used in phase contrast microscope.
Zernike discovered that if light rays are diffracted differentially, they can cause interference pattern in the image. Phase contrast microscope (PCM) has the ability to separate the direct light or incident light from the light diffracted by the specimen. Zernike developed a system of rings located both in the condenser lens and objective lens.
The light rays which arrive at the eye are out of phase and the image of specimen becomes enhanced. Colourless, transparent and invisible details of the specimen become visible PCM enhances the contrast of the transparent and colourless objects by influencing the optical path of light, causing difference in brightness. Invisible objects can be seen. It is a contrast enhancing optical technique.
For this purpose, PCM has two optical attachments. The first one is a diaphragm (D), which has an annular ring at the front focal plane of the condenser. It allows only a ring of light to pass through. The second is a phase-shifting plate (P) at the rear focal plane of the objective. It is transparent disc with an annular groove which coincides exactly with the image of the diaphragm. All the direct light now passes through the groove in the phase plate whereas the differenced light passes mainly outside the groove of the phase plate.
If the phase plate is made to retard the incident wave by a quarter of wavelength (1/4λ) the crest and though of the two waves will coincide, giving a resultant greater amplitude. Refractive details will appear brighter or dark.
As the diffracted light has to pass through a greater thickness of transparent glass material, a phase difference depending upon he refractive index of the phase place and the groove is created between direct and diffracted light rays. This altering of path of light rays is used to differentiate the different components of the cell or the specimen.
This is because different components of the specimen cause light rays deviate differentially or variably depending upon the density of each of them. It causes difference in brightness which enables us to study the transparent and colourless components. The different components of the specimen appear in various shades of gray.
PCM has made it possible to study living cells without staining, study of cell division, cell cycle, bacteria, sperms, tissues and sections etc.
