Transmission Electron Microscopy: Preparation of Specimens

Abstract

Sample preparation of biological specimens for electron microscopy aims at bringing the sample into a suitable size (<500 nm thick, accommodated on a 3 mm diameter, round sample carrier) and to strengthen these samples against the adverse conditions in the electron microscope (beam damage and vacuum). There are two types of samples, one are large cellular structures, which require further sectioning and the other are individual assemblies, which are small enough to be imaged in suspension. Any preparation method of these types of samples has to fulfil four basic requirements: (1) to avoid collapse of the structures in the vacuum of the electron microscope; (2) to provide a sample sufficiently thin to avoid multiple scattering of electrons; (3) to minimise structural alterations as a consequence of the damage by the electron beam; (4) to maximise the contrast in the resolution band of interest. The relative importance of each constraint varies with the type of specimen and with the level of resolution sought.

Key Concepts:

  • Fragile samples such as tissues, cells and some molecular machines are stabilised by cross‐linking with aldehydes (formaldehyde or glutaraldehyde) or Osmium tetroxide (lipids).

  • Vitrification by rapid freezing converts liquid water into amorphous solid water.

  • Vitrification perseveres ultra structures with the least artifacts (cryo fixation).

  • Vitrification is achieved by plunge freezing of thin objects (<1–2 μm) or by high pressure freezing of thicker objects (<600 μm).

  • Large samples such as cells and tissues are stabilised by embedding in plastic resins or by vitrification.

  • Thick objects are cut to appropriate thickness either with conventional ultramicrotomes (diamond knifes or glass knifes) or with a focused ion beam.

  • Contrast can be enhanced by staining procedures, which use electron dense heavy atom derivates such as Uranyl acetate, Phosphotungstic acid, osmium tetroxide, ammonium molybdate.

Keywords: electron microscopy; specimen preparation; transmission electron microscopy; sectioning; vitrification; negative staining; embedding; shadowing

Figure 1.

400 mesh copper grid held by a pair of tweezers. The grid has a diameter of 3 mm.The grid is somewhat translucent due to the holes and it has a wider rim (arrow) for handling.

Figure 2.

Schematic drawing of a negatively stained sample (perpendicular to the direction of imaging) and its projection image (shown in the direction of imaging) generated by the electron microscope. Top, the biological object (white circle) is supported by a carbon support film (brown line) and surrounded by the electron dense stain (black). The stain accumulates at the edges of the object and is less thick further away from the object. The electron microscope generates a projection of the object. Stain is excluded by the object. Thus these areas appear bright. Stain accumulates close to the object (giving a dark rim) and is less thick further away from the object. This gives rise to a density gradient around the object.

Figure 3.

Apparatus for vitrification. (a) Manual freezing apparatus with an environmental chamber. The humidification of the chamber is achieved with water soaked sponges. If hot water is used for soaking, the air inside the chamber is saturated with water. The grid is held by tweezers, which are mounted to a movable rod, which is accelerated by a spring (not visible) during plunging. The formation of a thin sample suspension is achieved by blotting with filter paper, which is mounted inside a chamber. A rod connects the filter paper to the outside and allows the user to handle it, without disturbing the humidity inside the chamber. The cryogen is placed in an insulating box outside the chamber. (b) Shows a fully computer controlled freezing apparatus. Blotting is done from both sides of the grid. The humidity inside the chamber is measured and adjusted with a nebuliser that generates small droplets of water with ultrasound.

Figure 4.

Viral capsids negatively stained (left) and vitrified (right). The negatively stained capsids appear bright against a darker background, because the stain in the background is more electron dense than the capsids. The contrast is high and the capsids can be easily recognised. The spikes of the capsid appear to have collapsed on the body of the capsids. In the vitrified sample (right) capsids are dark against a brighter background, because the protein is more electron dense than the vitrified buffer. The contrast is low, which makes it more difficult to recognise the particles. The spikes are better preserved giving the particles a more spherical appearance.

Figure 5.

Schematic drawing of unidirectional metal shadowing. The object is shown as a grey circle, the carbon support film as a brown line and the metal coat as a red line. The height of the object d can be calculated from the length of the shadow l and the shadowing angle α.

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Further Reading

Glauert AM and Lewis PR (1998). Biological Specimen Preparation for Transmission Electron Microscopy. In: Glauert AM (ed.) Practical Methods in Electron Microscopy, vol. 17. Princeton: Princeton University Press.

Griffiths G (1993) Fine Structure Immunocytochemistry. Berlin, Heidelberg and New York: Springer‐Verlag.

Jensen G (ed.) (2010) Methods in Enzymology Cryo‐EM, Part A Sample Preparation and Data Collection, vol. 481. Amsterdam and New York: Elsevier Science Publishing Co Inc.

Lewis PR and Knight DP (1977). Staining Methods for Sectioned Material. In: Glauert AM (ed.) Practical Methods in Electron Microscopy, vol. 5. Amsterdam, NY: Elsevier Science Publishing Co Inc.

Reid N and Beesley JE (1991). Sectioning and Cryosectioning for Electron Microscopy. In: Glauert AM (ed.) Practical Methods in Electron Microscopy, vol. 13. Amsterdam, NY: Elsevier Science Publishing Co Inc.

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Böttcher, Bettina(Jun 2012) Transmission Electron Microscopy: Preparation of Specimens. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002998.pub2]