Biomedical Engineering Reference
In-Depth Information
Box 2.2
continued
sheets of a solid inorganic scintillator which are
placed behind the film. Any emissions passing
through the photographic emulsion are absorbed
by the screen and converted to light, effectively
superimposing a photographic image upon the
direct autoradiographic image.
The gain in sensitivity which is achieved by use of
indirect autoradiography is offset by non-linearity
of film response. A single hit by a
b
-particle or
g
-ray
can produce hundreds of silver atoms, but a single
hit by a photon of light produces only a single silver
atom. Although two or more silver atoms in a silver
halide crystal are stable, a single silver atom is
unstable and reverts to a silver ion very rapidly.
This means that the probability of a second photon
being captured before the first silver atom has
reverted is greater for large amounts of radioactivity
than for small amounts. Hence small amounts of
radioactivity are under-represented with the use of
fluorography and intensifying screens. This problem
can be overcome by a combination of pre-exposing a
film to an instantaneous flash of light (pre-flashing)
and exposing the autoradiograph at
−
70°C.
Pre-flashing provides many of the silver halide
crystals of the film with a stable pair of silver atoms.
Lowering the temperature to
−
70°C increases the
stability of a single silver atom, increasing the time
available to capture a second photon (Fig. B2.2).
A
B
C
Fig. B2.2
The improvement in sensitivity of detection of
125
I-labelled IgG by autoradiography obtained by using an
intensifying screen and pre-flashed film. A, no screen and no pre-flashing; B, screen present but film not pre-flashed;
C, use of screen and pre-flashed film. (Photo courtesy of Amersham Pharmacia Biotech.)
