Electron Microscope
-Is a type of microscope that uses a beam of electrons to create an image of the specimen. It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail.
-In electron microscope, high-speed electron beam is 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 up to 1,000,000X(one million times). The Photographic techniques and computer aided techniques.
-The Electrons can be focused by electromagnetic 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 = 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.
Principles 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, tungsten is heated by applying a high voltage current, electrons form a continuous stream, which is used like a light beam.
•The lenses used in EM are magnetic coils capable of focusing the electron beam in 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.
How does it work?
•The light source is replaced by a beam of very fast moving electrons.
•The specimen usually has to be specially prepared and held inside a vacuum chamber from which the air has been pumped out (because electrons do not travel very far in air).
•The lenses are replaced by a series of coil-shaped electromagnets through which the electron beam travels. In an ordinary microscope, the glass lenses a bend (or refract) the light beams passing through them to produce magnification. In an electron microscope, the coils bend the electron beams the same way.
•The image is formed as a photograph (called an electron micrograph) or as an image on a TV screen.
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 disease 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 tumor identification, biopsy, study of cells, variety of molecules. In industry high resolution. 2D and 3D imaging. In forsenic, mining, chemical and petrochemical industries.
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 ssensor such as charge-coupled device (CCD) camera or 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.
-Instead of one picture of one section, series of sections are studied and analyzed. By computer aided averaging techniques of numerous image three dimensional reconstructions of cell organelles of highest clarity are developed. Tilting of specimen also provides three –dimensional picture.
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
The specimens are fixed in glutaraldehyde, osmium tetroxide 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
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, phosphotungstie acid. The thin sections soaked in solutions 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.
