Autoradiography

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.
History
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.
Radioisotopes,
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.
Detection
(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.
Autoradiography Method
 Living cells are briefly exposed to a ‘pulse’ of a specific radioactive compound.
 The tissue is left for a variable time.
 Samples are taken, fixed, and processed for light or electron microscopy.
 Sections are cut and overlaid with a thin film of photographic emulsion.
 Left 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).

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  The photographic emulsion is developed (as for conventional photography).
 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 (e.g. with Osmium on plastic
sections coated with stripping film [or dipping emulsion] as in papers by McGeachie and Grounds)
before exposure to the photographic emulsion. This avoids the need for individually (post-) staining
each slide.
 It is not necessary to coverslip these slides
 The position of the silver grains in the sample is observed by light or electron microscopy Note: the
grains are in a different plane of focus in the emulsion overlying the tissue section. Often oil with a x100
objective is used for detailed observation with the light microscope.
 These autoradiographs provide a permanent record.
 Full details on the batch of emulsion used, dates, exposure time and conditions should be kept for each
experiment.
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
 Wear gloves and work in the darkroom (using only a red safety light)
 Allow Kodak-NB2 emulsion (which comes as a thick white gel and is stored in the dark at 4oC) to come
to room temperature for 2 hours.
 Mix equal volumes of Kodak-NB2 and double distilled water together (say 5ml of each), place in a water
bath at 37oC and shake gently for about 15 mins.
 Dip slides vertically into a small amount of emulsion (about 2ml is all that is required) in a holder
designed to take one slide at a time (economises on the amount of emulsion)
 Place horizontally for about 15 minutes to air dry. Then stand vertically for at least 2 hours to dry.
 Transfer to a black, light free box and store in the fridge (4oC) with dessicant.
 Allow exposure time as specified e.g. 2 weeks. Or remove test slides at various times to determine
optimal exposure time for your particular situation.
Developing the film
 Use a dark room.
 Use Kodak D19 developer mixed 50:50 with water. Immerse slides for 4 mins.
 Wash in gently running tap water.
 Wash in double distilled water
 Use Ilford Hypan Rapid Fixer (leaflet T1812). Mix 40ml+160ml double distilled water +2ml Hypan
hardener. Immerse slides for 5mins.
 Wash in gently running tap water.

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  Note: once it is fixed can turn the lights on and tidy up. But make sure all sensitive film is put away
before you do this.
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:
 oxidative respiration,
 photosynthesis,
 the control of protein synthesis by nucleic acids,
 the timing of events throughout the cell cycle (Alberts p730),
 the fate of populations of cells - i.e number of cell divisions, migration, relationship of stem cells to the
final differentiated phenotypes (by tritiated thmyidine labelling of replicating cells - see lecture on Cell
Replication).
 Comparison of experimental treatments on events such as above
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:
 enzyme inhibitors,
 antibodies
 nucleic acid probes
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.

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