Lecture notes on autoradiography

Autoradiography is a technique, which uses several naturally occurring radioactive isotopes to trace the path by which materials move from one place to another within plants and animals. it is based on the ability to label cell components with radioisotopes)which can then be demonstrated by their capacity to interact with silver bromide crystals in a photographic emulsion. Now any biological compound can be labelled with radioactive isotopes opening up many possibilities in the study of living systems.
Principle
The mass of the atomic nuclei can vary slightly (= isotopes) for a particular element although the number of electrons remains constant and all the isotopes have the same chemical properties.the nuclei of radioactive isotopes are unstable and they disintegrate to produce new atoms and, at the same time, give off radiations such as electrons (beta rays) or radiations (gamma rays).)Naturally occurring radioisotopes are rare because of their instability, but radioactive atoms can be produced in nuclear reactors by bombardment of stable atoms with high— energy particles. The disintegrations can be detected in 3 ways. These detection methods are extremely sensitive and every radioactive atom that disintegrates can be detected.
Radiography is the visualization of the pattern of distribution of radiation. In general, the radiation consists of X-rays, gamma (Y) or beta (B ) rays, and the recording medium is a photographic film. In autoradiography the specimen itself is the source of the radiation, which originates from radioactive material incorporated into it. The recording medium which makes visible the resultant image is usually, though not always, photographic emulsion containing silver halide (e.g. AgBr) that is sensitive to the radioisotope.
Each crystal of silver halide in the photographic emulsion is an independent detector, insulated from the rest of the emulsion by a capsule of gelatin. Each crystal responds to the charged particle by the formation of a latent (hidden) image that is made permanent by the process of development. The record provided by the photographic emulsion is cumulative and spatially accurate. It provides information on the localization and distribution of radioactivity within a sample. Thus there is little point on doing autoradiography on a specimen that is homogeneously labelled. Autoradiography reveals the spatial difference in  distribution radioactivity within a specimen.
Autoradiography Procedure
1.Living cells are briefly exposed to a ‘pulse’ of a specific radioactive compound.
2.The tissue is left for a variable time to incorporate the labeled compound, which undergoes metabolic changes taking different routes within the cell.
3.Samples are taken, fixed, and processed for light or electron microscopy to detect the final location of the incorporated substance.
4.Sections are cut and coated with a thin film of photographic emulsion. This is done by holding the slide vertically and dipping in liquid (radiation-sensitive) emulsion contained in a jar.
5.Leave the specimen in the dark for days or weeks (while the radioisotope decays). This exposure time depends on the activity of the isotope, the temperature and the background radiation (this will produce with time a  contaminating increase in ‘background’ silver grains in the film).
6.The slides exposed to photographic emulsion are developed (as for conventional photography) to expose the dark silver grains of silver halide. Counterstaining e.g. with toluidine blue, shows the histological details of the tissue. The staining must be able to penetrate, but not have an adverse
affect on the emulsion. Alternatively, pre-staining of the entire block of tissue can be done.
7.The developed slides are then fixed in a suitable fixer to make them permanent.
8.The position of the silver grains in the sample is observed by light or electron microscopy.
9.These autoradiographs provide a permanent record.
10.If the tissue is exposed to the radioactive compound at successive intervals, the path of the compound within the cells can be traced.
Uses of Autoradiography

• Autoradiography is used to study the synthesis, turnover,transport and localization of micromolecular constituents in the cell.
• It has been used for studying the kinetics of DNA, RNA, proteins and carbohydrate synthesis. For example, in the classical experiment of Hershey, and Chase it vvas used toprove that  DNA is the genetic material.
• In molecular biology experiments, S35 and P32 are widely used to label nucleic acid probes to detect mRNA  by in situ hybridisation on tissue sections.
• Radioactive labelling of various molecules enables the binding of these molecules (as markers of other molecules) to be accurately monitored by radioisotope cytochemistry. e.g: enzyme inhibitors, antibodies, nucleic acid probes.
• Radioactive isotopes are also used to track the distribution and retention of ingested materials.