It uses magnetic fields to focus electron beams and produces enlarged image of minute object. It is a boon for microbiologists, which marked a significant improvement over light microscope. E. Ruska in 1931, developed electron microscope for which he received Noble Prize in physics in 1936. The wavelength of visible light limits resolution of light microscope. It is 200 nm (0.2 um). The observation of objects smaller than 200 nm needs electron microscope. The resolution of electron microscope is 0.5 nm. It uses electron beam of very short wavelength 0.005 nm.
Electron microscopes are of two types :
1. Transmission electron microscope (TEM)
2. Scanning electron microscope ( SEM)
1. Transmission electron microscope (TEM) :
The microscope in which an electron beam passing through a very thin (< 50 nm) metallized specimen forms an image on florescent screen called transmission electron microscope.
Principle :
Electron microscope works on the principle similar to that of light microscope. The circular magnetic lenses focus electron beams and produce magnified image of minute object. The magnetic objective produces primary magnified image. Magnetic intermediate projector lens further magnifies the primary image and produces final image.
Construction :
Transmission electron microscope consists of :
1. Electron gun : The electron gun is at the top of central column. The electron gun generates electrons from hot tungsten filament (cathode). A metal cylinder surrounds the tungsten filament known as Whenalt cap. This cap gives shape to the electron beam. Just below the Whenalt cap, electron gun contains an anode. The anode has a small hole at center through which electron beam passes. A very high voltage (30 to 150 KV) application between cathode and anode accelerates electrons at very high speed through the hole and down the column. The wavelength of electron beam is inversely proportional to the velocity. Here wavelength of radiation is 0.005nm.
2. Central column : It is an evacuated metal tube. Column maintains high vacuum to obtain clear image because air molecule deflect electrons. It consists of magnetic lenses and specimen holder.
a. Magnetic lenses : Electron microscope utilizes electromagnets called magnetic lenses to focus electron beams. Magnetic lenses consists of a coil of coils to produce a magnetic field. Focal length and magnification of magnetic lens changes by varying current. Following magnetic lenses are used:
i. Magnetic condenser lens : It focuses nearly parallel beam of electrons on the specimen.
ii. Magnetic objective lens : It forms a real primary image. It magnifies the image.
iii. Magnetic intermediate lens : It lies between objective lens and projector lens. It mangifies the primary image.
iv. Magnetic projector lens : It further magnifies a portion of primary image and projects image onto a fluorescent screen or photographic film.
b. Specimen holder : Electron microscope uses a thin, electron transparent, collodion membrane to prepare specimen for examination. A copper grid supports the membrane. A special holder holds the grid and introduces it into object plane through an air lock.
3. Fluorescent screen : The electrons harm eyes. The image is made visible by allowing it to strike a fluorescent screen (phosphorescent screen similar to the front of a cathode ray tube in a television set). To make permanent record image can be projected on a photographic film in a camera beneath the screen. The photographic image is further magnified four to six times giving magnification up to 10,00,000 times.
4. Viewing window : At the lower end of the column viewing window is present. Image on the fluorescent screen is viewed through the window.
Working
The working of electron microscope can be understood by comparing it with a light microscope upside down. The electron gun generates electrons form hot tungsten filament (cathode). A very high voltage (30 to 150 kV) application between cathode and anode accelerates electron at very high speed through the hole and down the column. Magnetic condenser lens focuses nearly parallel beam of electrons on the specimen. Magnetic objective lens forms a real primary image. Magnetic intermediate lens lies between objective lens and projector lens. It magnifies the primary image. Magnetic projector lens further magnifies a portion of primary image and projects image onto a fluorescent screen or photographic film.
The contrast in a transmission electron microscope results from differential scattering of electrons by the molecules of the specimen. Differential scattering of electrons by the molecules of the specimen leaves a shadow in the electron beam. A denser part in the specimen scatters more electrons and thus looks darker part in the image since fewer electrons strike that part of the screen whereas electron transparent areas appear brighter. Biological material composed mostly of atoms with low atomic number (C, H, O and N) cause less electron scatter and gives poor contrast.
Staining the specimen with heavy metal salts such as lead citrate and uranyl acetate can increase the contrast. These may be either fixed on the specimen (positive staining) or used to stain background (negative staining). Examination of viruses, bacterial flagella and protein molecules needs negative staining. Examination of internal fine structures of microorganisms requires thin sections (< than 50 nm), which stained positively. Microorganisms are fixed with acetone and soaked in liquid epoxy plastic. The plastic is allowed to form a solid block which is then cut by ultramicrotome.
2. Scanning electron microscope (SEM) :
The microscope in which an electron beam scans thick specimen and electrons emitted from an object's surface forms an image on fluorescent screen called scanning electron microscope .
It is used to study surface topography and morphology.
Application of electron microscope :
1. Observation of viruses.
2. To study bacterial flagella.
3. Detailed observation of intracellular structure.
4. Measurement of size of viruses.
5. Localization of enzymes.
6. Determination of size and shape of particles.
7. Determination of concentration of colloidal particles in suspension.