7.12.
AUTORADIOGRAPHY
255
that one apparently can know the position of the original latent image
with an error estimated to be approximately 200 A. Unfortunately it is
difficult to control these developers, so that success cannot be assured,
and their use is contraindicated for the present except when the highest
possible resolution is required. Hay and Revel (1963) are content to use
Dektol diluted with 2 parts water for development, and their silver grains
do not look notably different from those of Caro and van Tubergen.
Care should be taken not to overdevelop autoradiographic emulsions
in order to avoid the development of background grain, and to minimize
grain size. Thus, Hay and Revel use only a 2 minute development time
for grids for electron microscopy, whereas they use 5 minutes for
conventional microscopy. Relatively brief fixation also should be employed, for hyposulfite slowly dissolves silver.
It is of course well known that photographic grain size can be limited
by extreme underdevelopment. It might therefore be assumed that autoradiographic resolution could be substantially improved simply by
limiting the growth of silver grains, but this is not necessarily the case.
Meek and Moses (1963) have indeed produced punctate grains, only a
few hundred Angstroms in diameter, by short development times in cool
developers. However, one must assume that the limit of real resolution
is the initial size of the silver halide crystal in the undeveloped emulsion,
for it is thought that the reduction of the silver bromide to metallic silver
may begin anywhere within an exposed crystal, and not necessarily at
the exact point where it may have been struck by a (3-particle. Nothing is
really gained, then, by limiting the growth of silver grains to a size less
than the size of the original silver halide crystal.
Originally investigators attempted to study autoradiographed sections
with the gelatin of the emulsion still in place. Obviously this posed
serious handicaps since the thickness and density of the layer greatly
reduced contrast and electron microscopic resolution. It was subsequently
demonstrated that most if not all the gelatin could be removed without
losing or significantly altering the silver grains. Originally this was attempted with acid or alkali treatments, with proteolytic digestion, or
simply with warm water, but it has since been realized that the alkaline
lead hydroxide stains accomplish this quite effectively, while at the same
time advantageously staining the sections. While probably any of the
alkaline lead hydroxide stains may be used, Hay and Revel (1963)
specifically employed the Karnovsky Method A stain (Chapter 7.3),
AUTORADIOGRAPHY
255
that one apparently can know the position of the original latent image
with an error estimated to be approximately 200 A. Unfortunately it is
difficult to control these developers, so that success cannot be assured,
and their use is contraindicated for the present except when the highest
possible resolution is required. Hay and Revel (1963) are content to use
Dektol diluted with 2 parts water for development, and their silver grains
do not look notably different from those of Caro and van Tubergen.
Care should be taken not to overdevelop autoradiographic emulsions
in order to avoid the development of background grain, and to minimize
grain size. Thus, Hay and Revel use only a 2 minute development time
for grids for electron microscopy, whereas they use 5 minutes for
conventional microscopy. Relatively brief fixation also should be employed, for hyposulfite slowly dissolves silver.
It is of course well known that photographic grain size can be limited
by extreme underdevelopment. It might therefore be assumed that autoradiographic resolution could be substantially improved simply by
limiting the growth of silver grains, but this is not necessarily the case.
Meek and Moses (1963) have indeed produced punctate grains, only a
few hundred Angstroms in diameter, by short development times in cool
developers. However, one must assume that the limit of real resolution
is the initial size of the silver halide crystal in the undeveloped emulsion,
for it is thought that the reduction of the silver bromide to metallic silver
may begin anywhere within an exposed crystal, and not necessarily at
the exact point where it may have been struck by a (3-particle. Nothing is
really gained, then, by limiting the growth of silver grains to a size less
than the size of the original silver halide crystal.
Originally investigators attempted to study autoradiographed sections
with the gelatin of the emulsion still in place. Obviously this posed
serious handicaps since the thickness and density of the layer greatly
reduced contrast and electron microscopic resolution. It was subsequently
demonstrated that most if not all the gelatin could be removed without
losing or significantly altering the silver grains. Originally this was attempted with acid or alkali treatments, with proteolytic digestion, or
simply with warm water, but it has since been realized that the alkaline
lead hydroxide stains accomplish this quite effectively, while at the same
time advantageously staining the sections. While probably any of the
alkaline lead hydroxide stains may be used, Hay and Revel (1963)
specifically employed the Karnovsky Method A stain (Chapter 7.3),