Biological Sample Preparation and Observation Using Electron Microscopes
Resolution of a microscope depends on the wavelength of the light used. The electron microscope, which emerged in 1933, employs electron beams with a much shorter wavelength than visible light as its light source. This greatly improves its resolution compared with optical microscopes. Differences in light sources also lead to a series of distinctions between electron microscopes and optical microscopes.
Based on differences in electron beam imaging principles and ways the beam interacts with samples, modern electron microscopes have evolved into many types. The most commonly used ones are transmission electron microscopes (TEM) and scanning electron microscopes (SEM). The total magnification of TEM can range from 1000 to 1,000,000 times, while that of SEM ranges from 20 to 300,000 times. This experiment mainly introduces sample preparation for transmission electron microscopes and scanning electron microscopes.
II. Apparatus and Materials
Strain: Escherichia coli slant culture
Solutions and reagents: Formvar, concentrated sulfuric acid, absolute ethanol, sterile water, 2% aqueous sodium phosphotungstate solution (pH 6.5–8.0), 0.3% polyvinyl formal solution (dissolved in chloroform), cytochrome C, ammonium acetate, plasmid pBR322.
Instruments and supplies: Conventional optical microscope, copper grids, Buchner funnel, beaker, petri dish, sterile dropper, sterile forceps, dissecting needle, glass slide, hemocytometer, vacuum evaporator, critical-point dryer, etc.
III. Operating Procedures
(1) Sample Preparation and Observation for Transmission Electron Microscopy
Treatment of metal grids Samples for optical microscopy are observed on glass slides. However, for transmission electron microscopy, electrons cannot penetrate glass, so mesh substrates, commonly known as specimen grids, are used instead. Specimen grids are available in various specifications according to material and shape. Copper grids of 200–400 mesh are the most widely used. Copper grids must be cleaned before use to remove contaminants, otherwise the quality of supporting films and the clarity of specimen micrographs will be affected. This experiment adopts 400-mesh copper grids, treated as follows: first soak in Formvar solution for several hours, rinse repeatedly with distilled water, then dehydrate by soaking in absolute ethanol. If the grids remain dirty after the above steps, soak them in diluted concentrated sulfuric acid (1:1) for 1–2 minutes, or boil them in 1% NaOH solution for several minutes. Rinse thoroughly with distilled water, dehydrate in absolute ethanol, and set aside for later use.
