References: Alberts, p174-176, 730.
Rogers A (1979) Techniques of autoradiography. 3rd Edition. Elsevier, North Holland pp429.
Radiography is the visualisation of the pattern of distribution of radiation. In general, the radiation consists of X-rays, gamma (g ) or beta (b ) rays, and the recording medium is a photographic film. For classical X-rays, the specimen to be examined is placed between the source of radiation and the film, and the absorption and scattering of radiation by the specimen produces its image on the film. In contrast, 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.
The first autoradiography was obtained accidently around 1867 when a blackening was produced on emulsions of silver chloride and iodide by uranium salts. Such studies and the work of the Curies in 1898 demonstrated autoradiography before, and contributed directly to, the discovery of radioactivity. The development of autoradiography as a biological technique really started to happen after World war II with the development of photographic emulsions and then stripping film (see Rogers, 1979) made of silver halide. Radioactivity is now no longer the property of a few rare elements of minor biological interest (such as radium, thorium or uranium) as now any biological compound can be labelled with radioactive isotopes opening up many possibilities in the study of living systems.
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 (b rays) or radiations (g rays). Naturally occurring radioisotopes are rare because of their instability, but radioactive atom 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.
(i) Electrical: This depends on the production of ion pairs by the emitted radiation to give an electrical signal that can be amplified and registered: used in Geiger counter, ionisation counter and gas flow counter
(ii) Scintillation: Some materials have the property of absorbing energy from the radiation and re-emitting this in the form of visible light. In a scintillation counter these small flashes of light are converted into electrical impulses. Both of these techniques count the pulses of the disintegrating atoms. They are fast and quantitative.
(iii) Autoradiography differs from the pulse-counting techniques in several ways. 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 localisation and distribution of radioactivity within a sample (i & ii do not do this). Thus there is little point on doing autoradiography on a specimen that is homogeneously labelled. Although it can be quantitative, autoradiography is a much slower and more difficult approach.
Nuclear emulsions have a very high efficiency for b particles (electrons of nuclear origin), particularly those with low energies. Many of the isotopes of interest to biologists have suitable isotopes, e.g. tritium (= hydrogen-3), carbon-14, , sulphur-35 and iodine-125. The effective volume of the detector emulsion in the immediate vicinity of the source may be as little as 100 cubic microns.
Types of photograhic detection systems
Stripping film consists of an even layer of photographic emulsion on a supporting gelatin membrane (e.g. Kodak AR10), it is floated on water and then wrapped around the slide and forms very close contact as it dries (Rogers, Chap 15). This was once widely used but is now no longer made. It has the major advantage of uniform thickness but the disadvantage that the supporting membrane prevents counterstaining of the section and therefore the tissue block must be pre-stained before sections are coated.
Liquid photographic emulsion. This is the method routinely used today (see details below). It is simpler and much quicker to do, but the layer of liquid emulsion (e.g. Kodak NB2) can be slightly uneven in thickness as it flows down to the bottom of the slide as it is withdrawn: for most purposes this slight variation is not important, unless the number of grains are being strictly counted and compared across one slide (Rogers, Chap 16).
Method for coating and developing dipping emulsion
Coating the slides
Developing the film
X-ray film This is still widely used for macroscopic analysis of big specimens (not requiring a microscope). This film has much bigger crystal diameters and comes on hard sheets. It is traditionally used for analysing gels where the separated proteins or nucleic acids are labelled with radioisotopes.
Phosphoimager screen This is a new variation on detection of bands in radioactively labelled gels This has (i) very high sensitivity, (ii) a shorter development time and (iii) a major advantage is that the amount of signal gives a linear increase over a wide range of labelling intensities making quantitation very easy. Radioactive signal activates fluorescence in the screen (nothing is visible = latent image). The screen is scanned on a special densitometer, hooked to a computer which produces a digital picture. Can enhance the image and quantify the intensity of the signal. Can easily clear the screen and re-use.
Radioisotopes are used to trace molecules in cells and organisms
Tracer studies: Radioisotope labelling is uniquely valuable as a way to distinguish between molecules that are chemically identical but have different histories - for example those that differ in their time of synthesis. The earliest uses of autoradiography were for tracer studies e.g. radioactivity was used to label various molecules such as amino acids and then the way they were assembled into proteins over time throughout the cell could be followed. This technique was essential to understand:
Pulse chase is used to sharpen the resolution of timing in many of these experiments.
Analytical techniques: 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:
In molecular biology experiments , S 35 P 32 (and I 125) are widely used to label nucleic acid probes to detect mRNA by in situ hybridisation on tissue sections and also for quantitation by Northern analysis on gels. Radioisotope labelling has great sensitivity but the disadvantage that each time a hybridisation is performed, the probe has to be labelled with fresh radioisotope (since it decays rapidly) and this can be tedious and expensive. Furthermore radioisotopes are dangerous (especially I 125 ). For these reasons digoxygenin is now often favoured for labelling probes for in situ hybridisation studies (it is detected by an antibody and a colour reaction), particularly since digoxygenin-labelled probe is stable for many months.
Ingestion: Radioactive isotopes are also used to track the distribution and retention of ingested materials. Exotic radioisotopes with very short half-lives are used clinically ª