AUTORADIOGRA
PHY
D R . R U C H I K A YA D U
INTRODUCTION
•Autoradiography is the bio-analytical technique used to
visualize the distribution of radioactive labeled substance
with radioisotope in a biological sample.
• It is a method by which a radioactive material can be
localized within a particular tissue, cell, cell organelles or
even biomolecules.
• It is a very sensitive technique and is being used in a wide
variety of biological experiments.
• Autoradiography, although used to locate the radioactive
substances, it can also be used for quantitative estimation by
using densitometer.
HISTORY
• The first autoradiography was obtained accidently around 1867
when a blackening was produced on emulsions of silver chloride and
iodide by uranium salts observed by Niepce de St.Victor.
• In 1924 first biological experiment involving autoradiography traced
the distribution of polonium in biological specimens.
• 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 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 labeled with radioactive isotopes
opening up many possibilities in the study of living systems.
PRINCIPLE
• Autoradiography is based upon the ability of radioactive substance to
expose the photographic film by ionizing it.
• In this technique a radioactive substance is put in direct contact with
a thick layer of a photographic emulsion (thickness of 5-50 mm)
having gelatin substances and silver halide crystals.
• This emulsion differs from the standard photographic film in terms of
having higher ratio of silver halide to gelatin and small size of grain.
• It is then left in dark for several days for proper exposure.
• The silver halide crystals are exposed to the radiation which
chemically
converts silver halide into metallic silver (reduced) giving a dark color
band.
• The resulting radiography is viewed by electron microscope, preflashed
screen, intensifying screen, electrophoresis, digital scanners etc.
METHODOLOGY 1. The radioactive sample is
coveredwith the
photographic
emulsion
2. The radioactive part of
the sample activates the silver
halide crystals near by.
3. This results in reduction
of Ag+ ions to Ag atom
leaving dark color bands.
4. The slide is then washed
away by fixers to get
insoluble Ag atom only.
5.The autoradiogram can
further be viewed and
observed under the
microscope.
http://lifeofplant.blogspot.com/2011/12/autoradiography.html
BASIC MECHANISM
• Penetration of negatively charged beta particles emitted by
radioactive salts through silver halide film emulsion causes activation
of silver present in the emulsion.
• Activated silver crystals are very unstable therefore quickly reduced
to black silver particles which is easily detectable.
• Autoradiography sensitivity is improved by carrying the detection
process at 70°C and preflashing the film before use.
• Preflashing needs only one hit per crystal deposited to increases
sensitivity
Sequential steps of
autoradiography
Brief exposure of living cells to a pulse of specific
radioactive material for a variable
Dissection of samples into sections for coverage with thin film of
photographic emulsion which are
Development then incubated in the dark for
of photographic
few days for radioactive
emulsion decay.The exposure time depends on
Preparation of samples are for microscopy
isotope activity,
either light or electron
Toluidine blue is used for counter staining to reveal tissue histology.
Instead Osmium or dipping emulsion can be used for pre-
staining of the entire tissue before exposure to the photographic
emulsionMicroscopy
to avoid foreither light or
individual electron
post- is used
staining to slide.
each
determine the relative position of the silver
Generation of records in the form of
autoradiographs
CAIRNS’ TECHNIQUE FOR MEASURING
THE LENGTH OF DNA MOLECULES BY
AUTORADIOGRAPHY
1. Cells are grown in a solution containing radioactive thymidine
(tritiated thymidine – 3H-T)
2. The tritiated thymidine is incorporated into the chromosomal DNA of
the cell (3H-T is used as thymidine is not present in RNA)
3. The chromosomes are isolated by gently lysing the cells and
fixing the chromosomes to a photographic surface
4. The surface is then immersed in a radioactively-sensitive emulsion
containing silver bromide (AgBr)
5. The radiation released from the tritiated thymidine converts the
Ag+ ions in silver bromide into insoluble metal grains
6. Following a period of exposure, excess silver bromide is washed
away, leaving the silver grains to appear as small black dots
7. When the photographic film is developed, the chromosomal
DNA can be visualised with an electron microscope
FACTORS AFFECTING
EFFICIENCY OF
AUTORADIOGRAPHY
• 1. Energy of emitter: Higher the energy longer is the track length
and so it’s difficult to localize the points in the low density region of
the same track. Further very low energy radiation also creates a
poorer resolution image on the film. Therefore weak b-emitting
isotopes (3H, 14C and 35S) are most suitable because the energy of
radiation is in between g and a radiations.
• 2. Distance and Thickness of sample : If either the sample is very
thick or the sample is far away from the emulsion film, resolution will be
lost.
• 3. Grain size and amount of silver halide crystals : The grain
size should be smaller so that there is more availability of AgX
crystals. Also concentration of gelatin should be less in emulsion as
comapred to AgX crystals.
• 4.Thickness of emulsion:The emulsion thickness affects the
efficiency of autoradiography with different emitters. For b-emitters
the thickness of the emulsion should be less.
• 5. Exposure time : An autoradiogram must be exposed for a
sufficiently long time for proper exposure to view pattern of the
track length.
ADVANTAGES
• Technically easy not much expertise required,
• Highly specific detection tool,
• Unlike tissue bath preparations, pharmacologically characterize and localize
receptors in tissues,
• Enables characterization of receptors in different tissues in different animals
or brain regions
DISADVANTAGES
• Lack of assessment criteria to determine whether the binding
site really corresponds to an actual receptor,
• Non-physiological significance of high affinity radiolabelled receptor,
• Non-specificity of ligands can easily cause misinterpretation of results
APPLICATIONS
Autoradiography provides qualitative as well as quantitative information
regarding a specimen. Some of the following applications of this
technique are given below:
• Autoradiography is used to determine receptor distribution and
localization
while studying neurodegenerative disorders.
• Application of autoradiography in electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets during
blotting.
• To study cytogenesis of the forebrain.
• Applications in radiopharmaceutical research.
• Applications in radioimmunoelectroosmophoresis to study viruses.
APPLICATIONS (CONTINUED)
• In imaging and analyzing rock porosity
• In matrix-assisted laser desorption/ionization mass spectrometric
imaging (MALDI-MSI), and secondary ion mass spectrometric
imaging (SIMS-MSI) for pharmaceutical discovery and
development
• In whole body imaging.
• Tool for genetic studies.
• For comparison of complex mixtures of proteins.
• Applications in microbial ecology.
• Determining gross absorption and utilization of foliar applied nutrients etc.
REFERENCES
• https://www.omicsonline.org/open-access/autoradiography-detection-and-
analysis-of- radioactive-entities-2155-6180-1000361.php?aid=93116
• https://www.slideshare.net/kskuldeep1995/autoradiography-ks
• http://ib.bioninja.com.au/standard-level/topic-3-genetics/32-chromosomes/
chromosome- size.html
