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The Aaron Klug Centre for Imaging and Analysis

Lecture 2: Specimen preparation

Embedding, sectioning, negative staining and shadowing

animal sectionIn the study of biological material you will encounter material which is sufficiently finely divided to be examined directly e.g. Protein molecules, viruses, subcellular assemblies, liposomes etc and larger material. The dividing line is not sharp and depends on the accelerating voltage of the microscope and the resolution level that is required for the insights sought. Typically the "larger material" is cells and tissues. For most purposes these are examined as sections. These sections must be cut thin enough to allow the penetration of electrons. This is typically around 200nm. Cutting such thin sections requires that the material be "hard" - which biological material is not - so that the structure is not destroyed by the cutting. There are two solutions to this problem - freezing, which we will examine in the next lecture, and embedding in a plastic resin. The end effect of our efforts is shown on the right - an image in which the various tissue components can be easily recognized. To get there requires us to undertake a multi-step process, entailing:

  • Fixation
  • Dehydration
  • Embedding
  • Sectioning and
  • Staining

The reasons for this process should be appreciated. Let us return to the issue of embedding in a plastic resin - several resins are used - the most common being various epoxies and acrylic methacrylates.

Shadowing

Epoxy

Epoxies

Epoxies

Methacrylate

Methacrylate

Both can be infiltrated into the tissue as monomers and polymerised once in the tissue. The hardness can be varied by changing the length and the hydrophobicity can be changed by the use of adducts making them extremely versatile.

However the monomers are hydrophobic - making it necessary to remove the water from the specimen prior to infiltration. This is done fairly easily by putting the tissue through an alcohol series, i.e. A gradually increasing concentration of ethanol, usually 10%, 20%, 40%, 80%, 100% and ultimately propylene oxide (if epoxy resins are to be used). The removal of water from biological specimens is devastating for its structure. The hydrophobic effect is responsible for the maintenance of membrane integrity and for the maintenance of protein structure. Removal of water reduces biological material to a doughy goop. To avoid this we introduce both intermolecular and intramolecular chemical crosslinks - this process is called fixation and is usually considered to be the first step in the preparation of samples for electron microscopy. The primary agents used for doing this are aldehydes (usually glutaraldehyde and formaldehyde) and osmium tetroxide.

Glutaraldehyde

Glutaraldehyde

Aldehydes react with uncharged amino groups. These are present in both proteins and DNA. The favourite aldehyde of electron microscopists is glutaraldehyde - this has potentially two aldehydes that can react, however it readily polymerises to form a backbone with a number of pendant aldehyde groups.One reaction that is possible is the simple formation of a Schiff base however detailed studies show that a variety of reactions occur. Reactions with formaldehyde lead to the formation of methylene bridges between neighbouring amino groups.

Polymerised Glutaraldehyde

Polymerised Glutaraldehyde

There are various considerations concerning the use of aldehydes -

  • Ensure the pH is above 7
  • Never use a buffer that contains primary amines
  • Formaldehyde perfuses in faster, glutaraldehyde produces a better meshwork of crosslinks.
  • It is particularly important to match the osmolarity of your tissue to avoid distortion.
  • Aldehydes do not fix lipid bilayers

The last point implies that the lipids will be dissolved out of the tissue by the alcohol dehydration unless further steps are taken. This step is "post-fixation" with osmium tetroxide. This crosslinks the unsaturated lipids. Nevertheless approximately 45% of the lipid content of the cell is removed during dehydration.

Osmium tetroxide

Osmium tetroxide

Warning!

After the dehydration step monomers of the chosen resin are perfused into the tissue. This involves placing the fixed tissue in a mould and soaking it in resin. Resins are viscous and therefore the concentration of monomer is increased during more than one change of a mixture of monomer and solvent. Ultimately the monomer is polymerised. Epoxies are polymerised by heat in the presence of catalysts, acrylic methacrylates by UV irradiation.

showing moulds

moulds

The purpose of the mould is to ensure that the specimen can be easily located for microtomy in the chuck of the ultramicrotome. It is necessary to locate the area of interest by examining survey sections stained with toluidine blue in the light microscope and the cutting face has to be trimmed prior to ultramicrotomy.

Reichert FCS Cryo Ultramicrotome

trimming block

The sections are cut with glass or diamond "knives". Glass knives have a limited lifetime and are prepared soon before use.

Knife maker

Knife maker

Big glass

Glass

Knife maker

Knife maker

The design of the ultramicrotome incorporates a number of features which make it possible to easily set up the cutting operation and control the thickness of the sections.

sectioning

Sectioning

A water-filled-trough is attached to the knife so that cut sections can easily be picked up on copper grids.

close up
picking up sections
close up
picking up sections

Because the elemental composition of tissue is predominantly carbon, nitrogen, oxygen and phosphorus, all of which have low electron scattering factors, it displays very little structure when viewed in the electron microscope. To appreciate what comes next you should know that the scattering of electrons by heavy elements leads to amplitude contrast in the image - how this happens is the subject of a later lecture. Now you need to know that the structure can be visualised by reacting specific groups - notably phosphoryl groups - with heavy atoms - notably uranium in the form of uranyl. This process is called staining - sections are floated on solutions of uranyl acetate and lead citrate. This works to reveal the structure because the different components of tissue react differentially with the stain. As the reactions leading to heavy metal deposition are generally dependent on phosphoryl groups - the tissue elements stained most heavily are phospholipid membranes and components rich in RNA and DNA including ribosomes and the nucleus. However a variety of reactions result in lead and uranyl deposition and structure can be visualized in almost all tissue as a result of differential staining.

Particulate specimens

The primary method of visualization of particulate material is called negative staining. In this method particles are allowed to attach themselves to a carbon film, a droplet of solution containing a heavy metal is then applied to the film, wicked off with filter paper and allowed to dry. The effect is to encase the particles in the heavy metal salt. The biological material appears light against a dark background of stain - hence the name negative stain.

Negative stain

This is an extremely simple and effective technique and often the structural information is accurately preserved at resolutions below 2.5nm. There is however very little to support the structure in the vertical direction and it may collapse or distort due to the forces experienced on drying. Fortunately this occurs sufficiently infrequently and the technique remains useful. The two most useful negative stains are uranyl acetate and methylamine tungstate - both used at a concentration of about 2%. Depending on the specimen it may be necessary to pay attention to the pH of the stain solution, it is not possible to use high concentrations of other salts. Some specimens - especially those containing DNA - do not negatively stain particularly well. Also because the negative stain usually embeds the particle, and what is seen is a projection of the structure, it is not possible to determine its hand. An alternative technique is shadowing with heavy metals. This is accomplished by placing the specimen in a vacuum chamber, heating the metal until it vaporizes and then recondenses on the specimen.

shadowing
belljar
shadowing

Some examples of negatively stained specimens, and shadowed specimens:

haemocyanin
negative stained phage
shadowedphage1
shadowed phage
shadowed fibrinogen