Electron microscope are the magnifying instruments that utilizes the beam of high speed electrons to illuminate the object. Electron microscopy was discovered for visualizing the ultra-structure of the microorganisms, cells etc. It gives high magnification (up to 1000000x) and incredibly high resolution (dependent upon the wavelength of radiations).
Electron microscopes use signals arising from the interaction of an electron beam with sample to obtain information about structure, morphology and composition.
It uses electron beam to form the image of the object and magnification is obtained by electromagnetic fields. However, in light microscopes, light waves are used to produce the image and magnification is obtained by a system of optical lenses.
The first transmission electron microscope was developed by Ernst Ruska and Max Knoll of Germany in 1931.
The general principle of electron microscope is analogous to light microscope with the difference being use of electron beams to analyze the specimen instead of visible light. Electron gun or white hot tungsten filament emits high velocity electrons when high voltage [(40,000- 10,00,000) volts] is passed through it. Electrons (negatively charged) pass through hole in anode forming an electron beam, which is focused by condenser lens onto specimen.
Electrons are differentially imparted by various structures. Some of the electrons are scattered and some are absorbed by atoms of specimen. Electrons which pass through specimen are focused with series of magnetic objective lens onto photographic plate or fluorescent screen on which electrons interact as like photons and generate an image of the specimen.
The lenses used 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.
It is not possible to view living materials in electron microscope. The specimen to be observed under electron microscope needs to be prepared in special way. Various steps involved in sample preparation includes:
- Fixation and Dehydration
Biological samples are usually fixed with glutaraldehyde (cross-links proteins) & treated with osmium tetraoxide (stabilizes lipid bilayer and proteins). The specimens are fixed stabilize the cell structure. After fixation, dehydration is carried out slowly with organic solvents like acetone and ethanol. Dehydration of specimen is necessary as electron beam can only be produced and focused in vacuum.
As electrons have very little penetration power, samples must be invaded in special plastic resins (araldite and epoxy). Samples are embedded in plastic resin & soaked in un-polymerized, liquid epoxy plastic until it is completely permeated. Then it is hardened to form a solid block.
To obtain extremely thin sections from this plastic block, Ultra-microtomes with diamond knife or glass knives are used to cut it into thin sections (0.05-1 µm).
Specimens are stained with salts of heavy metals such as lead, osmium, uranium, phosphotungstic acid etc. The thin sections soaked in solutions of heavy metals like lead citrate, uranyl acetate or osmium tetroxide is also used for staining.
Contrast in electron microscopy is dependent upon atomic number of atoms in sample. Higher the atomic number, better the contrast. Hence, to obtain more contrast, staining of samples are done. Different cellular compartments and structures stains differently with heavy metals.
Electron Microscope has become a boon for biological sciences and industry as it gives immense magnification and resolution which has opened new areas and scope of research in cellular and molecular biology.
They are used in study of microorganisms like bacteria, virus and other pathogens (for diagnosis and treatment of disease), as well as fields of medicine (tumor identification, biopsy), human anatomy (study of cells), pathology, forensic science, nanotechnology (variety of molecules and nano particles) etc. Microbiology has been greatly developed and modernized after development of electron microscope
It is also used in various industries for high resolution, 2D and 3D imaging, mining as well as chemical and petrochemical industries.
There are various advantages of electron microscopy over light microscopy as limitations in light microscopy gives rise to it.
- EM has a high magnification and resolving power.
- Study of submicroscopic cell organelles (e.g., ribosomes, micro-bodies, centrioles, microtubules, endoplasmic reticulum) and internal structure of microscopic organelles (e.g., chioroplasts, mitochondria.
- Study of microorganisms, viruses and virions.
- It can discern even macromolecules.
- Helps to know the arrangement of molecular aggregates and even their components, e.g. nucleosomes.
There are few drawbacks of using electron microscopy which are:
- Complicated and Expensive.
- Risk of radiation leak.
- Require very high voltage electric current.
- Need of cooling system.
- The specimen or object has to be given special treatment E.g. complete dehydration.
- Rigorous sample preparation.
- Living stage of microorganisms cannot be observed.
Types of Electron Microscope
There are two types of electron microscope
- Scanning electron microscopy
- Transmission electron microscopy.