Biomedical Engineering Reference
In-Depth Information
Box 2.2 The principles of autoradiography
The localization and recording of a radiolabel within
a solid specimen is known as autoradiography
and involves the production of an image in a
photographic emulsion. Such emulsions consist of
silver halide crystals suspended in a clear phase
composed mainly of gelatin. When a
b
-particle or
g
-ray from a radionuclide passes through the
emulsion, the silver ions are converted to silver atoms.
This results in a latent image being produced, which
is converted to a visible image when the image is
developed. Development is a system of amplification
in which the silver atoms cause the entire silver halide
crystal to be reduced to metallic silver. Unexposed
crystals are removed by dissolution in fixer, giving
an autoradiographic image which represents the
distribution of radiolabel in the original sample.
In direct autoradiography, the sample is placed
in intimate contact with the film and the radioactive
emissions produce black areas on the developed
autoradiograph. It is best suited to detection of
weak- to medium-strength
b
-emitting radionuclides
(
3
H,
14
C,
35
S). Direct autoradiography is not suited
to the detection of highly energetic
b
-particles,
such as those from
32
P, or for
g
-rays emitted from
isotopes like
125
I. These emissions pass through and
beyond the film, with the majority of the energy
being wasted. Both
32
P and
125
I are best detected
by indirect autoradiography.
Indirect autoradiography describes the technique
by which emitted energy is converted to light by
means of a scintillator, using fluorography or
intensifying screens. In fluorography the sample
is impregnated with a liquid scintillator. The
radioactive emissions transfer their energy to the
scintillator molecules, which then emit photons which
expose the photographic emulsion. Fluorography
is mostly used to improve the detection of weak
b
-emitters (Fig. B2.1). Intensifying screens are
35
S
3
H
+
−
+
−
Fig. B2.1
Autoradiographs showing the detection of
35
S- and
3
H-labelled proteins in acrylamide gels with (
+
) and without
(
−
) fluorography. (Photo courtesy of Amersham Pharmacia Biotech.)
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