Preparative Techniques for the TEM
For routine transmission electron microscopy (TEM), it is generally accepted that specimens should be thin, dry and contain molecules which diffract electrons. Biological specimens, which are large and consist of large amounts of water, also do not defract electrons and are therefore difficult to see in the TEM. Preparing biological specimens for the TEM, whilst retaining the structural morphology of the material, is a challenge. However, researchers have been looking at biological material for many years, and many protocols exist which allow us to look at biological material in many different ways. Below is a brief outline of some of the more common ways of looking at biological samples in the TEM.
Small or very thin objects can be examined directly by mounting them onto a support film and introducing them directly into the electron beam. Contrast is provided by heavy metal precipitation in one of three ways.
- Positive staining: The object is chemically stained with a solution of the metal salt and appears dark on a bright background.
- Negative staining: The object remains unstained but is embedded in a dried film of the heavy metal salt. The specimen appears light on a dark background. This method of visualization has been used extensively in the study of virus particles but is also useful for cell fractions (e.g. coated vesicles). More details can be found here.
- Shadowing: A thin layer of heavy metal atoms is deposited on the specimen by evaporation in a vacuum chamber. Shadowing from one direction only produces a pseudo-three-dimensional image. Rotary shadowing, where the specimen is uniformly coated with heavy metal, is used to visualize nucleic acids and proteins.
The most popular technique for examining biological materials is to embed the material under study in plastic and cut ultrathin sections that can be examined in a TEM. The material is stabilized by chemical fixation (usually with aldehydes such as formaldehyde or gluteraldehyde), contrasted with solutions of heavy metal salts (osmium tetroxide and uranyl acetate), dehydrated in ethanol or acetone, and embedded in plastic (epoxy resin). Ultrathin sections (60 nm) cut with glass or diamond knives using an ultramicrotome are floated on water, transferred to specimen support grids and examined in the TEM. Often the sections are further contrasted with uranyl acetate and lead citrate prior to examination in the microscope.
In some cases, macromolecules can be specifically labelled prior to embedding and sectioning. For example, the location of some enzymes can be visualized by incubating the tissue with a substrate whose reaction with the enzyme leads to the local deposition of electron opaque material. Alternately, antibodies can be coupled to such enzymes, and the electron opaque reaction product is used to localize the antigens recognized by the antibodies. Some embedding resins (e.g. Lowicryl resins and LR White resin) have been designed to enable antibodies and electron opaque markers (such as colloidal gold particles) to be applied to the ultrathin sections. In this way, subcellular antigens recognized by the antibodies can be localized with the TEM.
Another sectioning technique that is increasing in popularity is cryosectioning (the sectioning of vitrified, frozen material). After chemical fixation, the tissue is immersed in cryo-protectant (usually sucrose) and then quickly frozen in liquid nitrogen. The cryo-protectant allows the biological material to be frozen without the formation of ice crystals, which would damage ultrastructure. This type of freezing, or vitrification, is possible in the absence of cryo-protectants but is technically demanding. Sections cut from the vitrified block can be thawed and incubated with antibodies specific to subcellular antigens. Electron opaque markers allow the antibodies to be seen in the TEM.
Colloidal gold coupled to protein A (a protein from bacterial cell walls which binds to the Fc portion of some immunoglobulins) has been used extensively in recent years to localize antibodies on resin and frozen sections of biological materials. The ability to produce homogeneous populations of colloidal gold with different particle sizes has enabled researchers to use these probes to colocalize different structures on the same section.
It is possible to freeze biological material fast enough to vitrify the water present inside the cells. Vitrification of water occurs when the freezing has occurred so fast that ice crystals have no time to form. Vitrified biological material can be sectioned at low temperatures. Thin films of vitrified water and sections of vitrified material can be examined in transmission electron microscopes that are equipped with specimen stages that can be kept cold.
Rapid Freezing Methods
There are seven main rapid freezing methods presently available. They are
- immersion freezing - the specimen is plunged into the cryogen.
- slam (or metal mirror) freezing - the specimen is impacted onto a polished metal surface cooled with liquid nitrogen or helium.
- cold block freezing - two cold, polished metal blocks attached to the jaws of a pair of pliers squeeze-freeze the specimen.
- spray freezing - a fine spray of sample in liquid suspension is shot into the cryogen (usually liquid propane).
- jet freezing - a jet of liquid cryogen is sprayed onto the specimen.
- high pressure freezing - freezing the specimen at high pressure to subcool the water.
- excision freezing - a cold needle is plunged into the specimen, simultaneously freezing and dissecting the sample.
Freeze-fracture followed by freeze etch and replication
If, for some reason, the object to be studied cannot be examined in the TEM, then a thin replica can be made. This is usually made by evaporating a thin layer of a heavy metal (usually platinum) onto the specimen and then coating this with a thin layer of carbon. The object and the replica are separated either by floating off the replica or by digesting away the object. There are four basic steps to follow
- The specimen is frozen (often without regard to vitrification).
- The specimen is fractured, while still frozen, under vacuum.
- The fractured specimen can then be etched by leaving it frozen and under vacuum. Depending on the time of exposure, more or less water sublimes from the specimen (freeze drying).
- A replica of the fractured surface is made which is then examined in the electron microscope.
A recent modification of this method employs rapid freezing achieved by slamming cells against a copper block cooled to -269°C with liquid helium. If these frozen cells are then exposed to extensive freeze drying (deep etching), very impressive images of the internal structures of cells are uncovered.