Photographic images are the final output of any electron microscopy project. These images can represent hours of sample processing, viewing, and photographing and will probably be the only results that most people see. It is therefore very important that the investigator understand the various aspects of EM photography in order to optimize the final images.
All modern electron microscopes are equipped with facilities for the recording of images produced on them. Essentially, they can be thought of as very specialized cameras.
General Steps in Producing an Electron Micrograph
Expose the sensitive photographic emulsion by means of electrons.
Components of Photographic Film
1) Emulsion
The electron/light sensitive emulsion is the part of the film that forms the actual image. The emulsion consists of a gelatin binder in which are suspended silver halide crystals, usually silver bromide. The gelatin keeps the siler halide crystals dispersed and facilitates the production of an even coating on the base material. Importantly, the gelatin also allows penetration of developer and other chemicals during processing. When dry, the emulsion is tough enough to stand up to reasonable handling but film should always be handled by the edges and with clean hands.2) Base Material
The base material must be strong and transparent. In the past, glass has been the choice for TEM film. It is very stable, but fragile and hard to store. Today, a polyester base material is commonly used in TEM sheet films where maximum dimensional stability is required. Roll films are often cellulose triacetate, a nonflammable substance (hence the term "Safety Film" - early films were made using cellulose nitrate, which proved to be unstable and highly flammable).An important consideration for TEM film is the "outgassing" of the base material. This is the amount of trapped gasses and water vapor in the film base that must be removed from the film to be stable in the high vacuum of the electron microscope. Modern plastic based EM film (such as Kodak "Estar" thick base film) combine ease of storage with the low out gassing and high stability of glass negatives.
3) Antihalation Layer
TEM films often have an antihalation layer which is a dye applied to the back of the film to prevent electrons from bouncing off the camera back and being reflected back through the emulsion. This produces "fog" (noninformational silver grains in the final image) and reduces resolution. The antihalation layer also acts to prevent scratching and curl of the film. Photographic paper has the same components as film with a base material of resin coated paper. There is no need for an antihilation layer because the paper is not transparent.4) Exposure
Exposure of a film occurs when electrons/photons interact with the silver halide crystals and reduce ionic silver in the silver halide crystals to metallic silver. Electrons are much more efficient than photons at reducing silver halide grains. One electron can reduce about 10 silver grains while 8-10 photons are required to reduce 1 silver grain.These interactions form a latent image where where a small free silver "speck" is released within the silver halide crystal. The latent image is a record of the portions of the emulsion that have been exposed to electrons. This image, however, is not visible and must be processd to produce a stable photographic image.
The size of the grains vary with the sensitivity (or spread) of the emulsion. Generally, the more sensitive (faster) emulsions have larger grains while less sensitive (slower) emulsions have finer grains and therefore better resolution so, there is a trade off between emulsion speed and resolution. The grains range in size roughly from 0.3 to 1 micron.
Electrons generated in the electron microscope have high energy and one electron can cause a latent image to form. As a result, emulsions on TEM film are much thicker (12µm) than film used for light photography.
