Preparation and Observation of Biological Samples for Electron Microscopes
The resolution of a microscope depends on the wavelength of the light used. The electron microscope, which emerged in 1933, achieved significantly higher resolution than an optical microscope due to the use of electron beams with wavelengths much shorter than visible light as light sources. The different light sources also determine a series of differences between electron microscopes and optical microscopes.
Based on the differences in the principles of electron beam imaging and the ways in which they act on samples, modern electron microscopes have developed into many types. Currently, the most commonly used are transmission electron microscopes and scanning electron microscopes. The former has a total magnification that can vary between 1000-10000000 times, while the latter has a total magnification that can vary between 20-300000 times. This experiment mainly introduces the preparation of two types of microscope samples, transmission electron microscopy and scanning electron microscopy.
2, Equipment
1. The strain Escherichia coli is inclined.
2. Solution or reagent: pentyl acetate, concentrated sulfuric acid, anhydrous ethanol, sterile water, 2% sodium phosphotungstate (pH 6.5-8.0) aqueous solution, 0.3% polyvinyl formaldehyde (dissolved in chloroform) solution, cytochrome c, ammonium acetate, plasmid pBR322.
3. Instruments or other tools include ordinary optical microscopes, copper nets, porcelain funnels, beakers, petri dishes, sterile droppers, sterile tweezers, pins, glass slides, counting plates, vacuum coating machines, critical point dryers, etc.
3, Operation steps
(1) Preparation and Observation of Samples for Transmission Electron Microscopy
1. Treatment of metal mesh
The sample of an optical microscope is placed on a glass slide for observation. However, in a transmission mirror, since electrons cannot penetrate the glass sheet, a mesh material can only be used as the carrier, commonly referred to as a carrier mesh. The carrier mesh can be divided into various specifications due to different materials and shapes, among which copper mesh with 200-400 mesh (number of holes) is commonly used. Copper mesh should be treated before use to remove any dirt and keep it clean, otherwise it will affect the quality of the supporting film and the clarity of the specimen photos. The 400 mesh copper mesh used in this experiment can be treated by the following method: first, soak and float with pentyl acetate for a few hours, then rinse several times with distilled water, and finally immerse and float the copper mesh in anhydrous ethanol for dehydration. If the copper mesh is still not clean after the above methods, it can be soaked in diluted concentrated sulfuric acid (1:1) for 1-2 minutes, or boiled in 1% NaOH solution for a few minutes, rinsed several times with distilled water, and then dehydrated in anhydrous ethanol for later use.
