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.