Preparation and Observation of Biological Specimens for Electron Microscopes
The resolution of a microscope depends on the wavelength of the light it uses. The electron microscope, which emerged in 1933, uses electron beams with a much shorter wavelength than visible light as its light source. Therefore, it achieves a far higher resolution than optical microscopes. The difference in light sources also brings about a series of distinctions between electron microscopes and optical microscopes.
Based on differences in electron beam imaging principles and ways of interacting with samples, modern electron microscopes have evolved into many types. The most widely used ones are transmission electron microscopes (TEM) and scanning electron microscopes (SEM). The total magnification of the former ranges from 1000 to 1,000,000 times, while that of the latter ranges from 20 to 300,000 times. This experiment mainly introduces sample preparation for transmission electron microscopes and scanning electron microscopes.
II. Apparatus and Reagents
① Bacterial strain: Escherichia coli slant culture. ② Solutions or reagents: amyl acetate, concentrated sulfuric acid, absolute ethanol, sterile water, 2% sodium phosphotungstate aqueous solution (pH 6.5–8.0), 0.3% polyvinyl formal (dissolved in chloroform) solution, cytochrome c, ammonium acetate, plasmid pBR322. ③ Instruments and supplies: ordinary optical microscope, copper grids, ceramic funnel, beaker, petri dish, sterile dropper, sterile forceps, dissecting needle, glass slide, counting chamber, vacuum evaporator, critical point dryer, etc.
III. Operating Procedures
(A) Sample Preparation and Observation for Transmission Electron Microscope
① Treatment of metal grids Samples for optical microscopy are observed on glass slides. However, for transmission electron microscopy, electrons cannot penetrate glass slides, so mesh materials called support grids are used as sample carriers. Support grids are available in various specifications according to material and shape; copper grids of 200–400 mesh are the most commonly used. Copper grids must be pre-cleaned before use to remove contaminants, otherwise the quality of the supporting film and the clarity of specimen micrographs will be affected. A 400-mesh copper grid is adopted in this experiment, and the treatment steps are as follows: first soak the copper grid in amyl acetate for several hours, rinse it several times with distilled water, then dehydrate it in absolute ethanol. If the copper grid is still dirty after the above treatment, soak it in diluted concentrated sulfuric acid (1:1) for 1–2 minutes, or boil it in 1% NaOH solution for several minutes. After rinsing repeatedly with distilled water, dehydrate it in absolute ethanol for standby use.
