Autoradiography

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AUTORADIOGRAPHY
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Radioisotopes

• Isotopes(which have same chemical but different
  physical properties) having radioactivity.
• Natural occurrence is rare because of its
  instability.
• They emit different radiations such as a,ß and ?.

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Detection of disintegration

• Electrical
• Eg GM counter, Ionisation
  counter, Gas flow counter
• Scintillation
• Eg scintillation counter
• Autoradiography

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What is autoradiography?
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Principle
Radiation will hit silver grains in emulsion and
expose them
Expose to film or emulsion
Isotope will emit radiation (usually beta)
Incubate tissue with radioactive ligand
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HISTORY

• 1867 Uranium salts responsible for blackening
• of photographic film(Niepce de
  st.victor)
• 1896 K2UO2(SO4)2emits radiation affecting a
• photographic film(Becquerel)
• 1924 First autoradiogram distribution of Po
  in
• biological material
• 1943 Autoradiographic demonstration of 131I
  in
• tissue sections of thyroid
• 1946 Liquid photographic emulsion for
• autoradiography(Belanger and
  LeBlond)
• 1950 Stripping film technique for
  microscopic
• autoradiography
• 1970 Numerous biological applications
• 1990 Alternatives to film Imaging plate
  technology

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PROCEDURE
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• Types
• In-vivo autoradiography
• In-vitro autoradiography

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Cont
MICROSCOPIC AUTORADIOGRAPHY
Hypophysis of rat labelled with 3H
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Macroscopic autoradiography
Localization on chromatogram or gel
Whale body autoradiography
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RADIONUCLIDES USED
Radionuclide Radiation Type of energies
125I eAU, eIC X,? 3-35Kev 3-35Kev
3H ß 18.5Kev
35S ß 167Kev
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• Localization
• Electron microscopical
• (cell and organelle level)

Thyroid of mouse after injection of 125I
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Light microsopical (cell and tissue level)
Hypophysis of rat labelled with 3H
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Macroscopical
Autoradiogram of a section through the head of a
monkey after intravenous injection af
35S-chlorpromazine.
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Quantification

• Grain counting
• Track counting
• Densitomety

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development

• X-ray film

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IP Scanner
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Imaging plate
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PHOTOGRAPHIC DETECTION SYSTEMS

• Stripping film
• Eg. Kodak AR10
• Liquid photographic emulsion
• Eg. Kodak NB2

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The Photographic Process
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2
3
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Special Types Of Autoradiography

• Fluorography

FILM
RADIOACTIVE OBIECT
ß-PARTICLE
LIGHT
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Autoradiography/fluorography and chromatography
Traditional autoradiography with X-ray
film ß-particles act directly on the film
Enhanced autoradiography chromatogram treated
with vax-based scintillator ß-particles absorbed
in the object energy converted into light,
object transparent
Fluorography chromatogram treated with PPO
ß-particles absorbed in the object energy
converted into light
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• Chemiluminescence

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• Advantages
• Highly specific tool to pharmacologically
  characterize receptors in tissue
• Provides location of receptor in tissue
• Enables characterization of receptors in
  different tissues between different animals or
  brain regions
• Technically easy

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Disadvantages

• Everything binds to everything (easy to
  misinterpret results)
• There are no biochemical or physiological
  criteria to
• assess the binding specificity (i.e., to
  determine whether the binding site really
  corresponds to an actual receptor)
• The presence of a high-affinity radiolabelled
  receptor does not necessarily imply that the
  receptor has physiological significance
• Ligands are not always very specific

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Autoradiographic investigation of protein
synthesis an example
Model based on pulse-chase experiments and
EM-autoradiography
Eucaryote cells in culture are subjected to a
pulse of radioactive amino acids, followed by a
surplus of unlabelled amino acid (chase). Cells
are drawn from the culture at different times
after the pulse, they are fixed and dyed for
electron micro-scopy, and exposed to
autoradiographic emulsion. After development and
fixation of the emulsion, the material is
investigated in the electron microscope. It is
possible to observe the cell organelles and the
location of radioactive material simultaneously.
The latter changes with time after the pulse.
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Autoradiographic investigation of cell kinetics
an example

• Eucaryote cells (in vivo or in vitro) are
  subjected to a pulse of 3H-thymidine, which is
  incorporated in DNA in the cells that are in
  S-phase during the pulse. The proportion of
  labelled cells in the population may be used for
  deriving parameters of cell kinetics (e.g. the
  duration of the cell cycle).
• In a variant of the technique, the cells
  are first labelled with 3H-thymidine, and
  subsequently after a known time interval ?t
  with 14C-thymidine. Due to the different range
  of the ß-radiation from 3H and 14C it is possible
  to distinguish between cells labelled with 3H,
  14C, or with both 3H or14C. The method provides
  information on additional cell kinetic
  parameters, e.g. the duration of the S-period.

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Autoradiographic mapping of CNSan example
binding af 3HHC3 in rat brain

• Localization of
• Receptors
• Transporters
• Etc.

• Autoradiographical localization of receptors and
  other specific sites in peripheral tissues or
  in the CNS advantages as compared to biochemical
  methods
• More accurate anatomical localization (at the
  neuron-level)
• High sensitivity (103104 higher than for
  biochemical assays)

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• Other Uses
• Map anatomical location of radiolabelled ligands
  to visualize and quantify receptors in tissue
• Trace neurons by axonal transport of
  radioactively labelled amino acids, certain
  sugars, or transmitter substances
• Measure DNA production (e.g., 3H-thymidine)
• Study of DNA viruses
• Ingestion Radioactive isotopes are also used to
  track the distribution and retention of ingested
  materials. Exotic radioisotopes with very short
  half-lives are used clinically

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CONCLUSION
Autoradiography can help to
establish the presence of radioactive elements in
various ores, radioactive material in tissues of
organisms. Makes it possible to gather accurate
data describing in which cell processes takes
place . Various substances are localized in the
tissue using this technique. This method also
helps in in establishing time parameters for
various process.
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THANK YOU