Preparation and observation of biological samples for electron microscopy
The resolution of a microscope depends on the wavelength of the light used. The electron microscope, which began to appear in 1933, uses an electron beam with a wavelength much shorter than that of visible light as a light source, so that the resolution it can achieve is greatly improved compared to that of an optical microscope. The difference in light sources also determines a series of differences between electron microscopes and optical microscopes.
According to the differences in the principles of electron beam imaging and the different ways of acting on samples, modern electron microscopes have developed into many types. Currently, the most commonly used ones are transmission electron microscopes and scanning electron microscopes. The total magnification of the former can be between 1000-1000000. The total magnification of the latter can vary between 20 and 300,000 times. This experiment mainly introduces the preparation of two types of microscope samples, transmission electron microscope and scanning electron microscope.
2. Equipment
1. Bacteria Escherichia coli (Escherichia coli) slant.
2. Solution or reagent amyl acetate, concentrated sulfuric acid, absolute ethanol, sterile water, 2% sodium phosphotungstate (pH 6.5-8.0) aqueous solution, 0.3% polyethylene formaldehyde (soluble in chloroform) solution, cells Pigment c, ammonium acetate, plasmid pBR322.
3. Instruments or other utensils: ordinary optical microscope, copper mesh, porcelain funnel, beaker, dish, sterile dropper, sterile tweezers, pins, slides, counting board, vacuum coating machine, critical point dryer, etc.
3. Operation steps
(1) Sample preparation and observation for transmission electron microscopy
1. Treatment of metal mesh
The sample for optical microscopy is placed on a glass slide for observation. However, in transmission electron microscopy, since electrons cannot penetrate the glass sheet, mesh materials can only be used as carriers, usually called carrier nets. The carrier mesh can be divided into many different specifications due to different materials and shapes, among which the most commonly used is 200-400 mesh (number of holes) copper mesh. The copper mesh must be treated before use to remove dirt and keep it clean, otherwise it will affect the quality of the support film and the clarity of the specimen photos. This experiment uses a 400-mesh copper mesh, which can be treated as follows: first, soak and bleach it with amyl acetate for several hours, then rinse it with distilled water several times, and then soak it in absolute ethanol for dehydration. If the copper mesh is still not clean after the above methods, you can soak it in dilute concentrated sulfuric acid (1:1) for 1 to 2 minutes, or boil it in 1% NaOH solution for a few minutes, rinse it with distilled water several times, and then put it in anhydrous Dehydrate in ethanol and set aside.
2. Preparation of support membrane
When observing samples, the carrier net should also be covered with a layer of unstructured, uniform film, otherwise small samples will leak out from the holes of the carrier net. This film is usually called a support film or carrier film. The support film should be electron transparent, and its thickness should generally be less than 20nm; under the impact of the electron beam, the film should also have a certain mechanical strength to maintain structural stability and have good thermal conductivity; in addition, the support network should be used in electron microscopy There should be no visible structure underneath, no chemical reaction with the sample being carried, and no interference with the observation of the sample. Its thickness is generally about 15nm. The supporting film can be plastic film (such as collodion film, polyethylene formaldehyde film, etc.), carbon film or metal film (such as beryllium film, etc.). Under normal working conditions, plastic film can meet the requirements. Among plastic films, collodion film is relatively easy to prepare, but its strength is not as good as polyethylene formaldehyde film.
