Electron microscopes produce an image of a specimen by using a beam of electrons rather than a beam of light. As electrons have shorter wavelength than visible light, electron microscopes to produce higher-resolution images. It 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).
Types of electron microscopy scanning and transmission electron microscopes.
Sample Preparation in Electron microscopy
Even though there are advantages of electron microscopes over the light microscopes, sample preparation requires a special process. As it is not possible to view living materials through electron microscopy, 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. Neutral formalin or alcohol formalin can also be used instead of glutaraldehyde.
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 unpolymerized, liquid epoxy plastic until it is completely permeated. Then it is hardened to form a solid block. Embedding medium is a mixture of equal parts of n-butyl and ethyl methacrylate polymerized by ultra-violet light.
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). Sections must be very thin because electrons of the standard electron microscope cannot pass through biological material much thicker than 150 nm. For best resolutions, sections should be from 30 to 60 nm.
Although it is used mostly for biological specimens but plastics and soft metals sections can also be prepared.
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.
- Sections are dried onto a slide and stained with either 75% methylene blue and 0.25% azure B or 0.5% methylene blue and 0.5% azure II in 0.5% aqueous borax and heated over a flame for 8–10 sec.
- The slides are rinsed with water, then stained the same way with 0.1% basic fuchsine in 5% aqueous ethanol.
The stained cytoplasm appears blue, nuclei darker blue, fat and intracellular lipid droplets gray-green, collagen, mucus and elastin pink to red.
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.