Autoradigraphy, In vivo autoradiography, In vitro autoradiography, SPECT, radiolabelled p32 and C14,

1. AUTORADIOGRAPHY
Dr. M. THIPPESWAMY

2. 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.

3. 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

4. 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 (AgX – silver bromide, chloride, iodide
or fluoride – AgBr, AgCl, AgI or AgF, respectively).
• 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.

5. METHODOLOGY
1. The radioactive sample is covered with
the photographic emulsion by several
described method.
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.

6. A silver halide (AgX) is ionized by the radiation emitted from
radioisotopes, forming Ag+ ions.
Ag+ is then reduced and converted to metallic Ag by a developer reagent
(usually containing AgNO3), which precipitates within the gelatin emulsion
of the X-ray film.
The reduction and precipitation are stopped by emerging the film in a
fixative solution forming the final image.
General principle of autoradiography

7. Autoradiography: Methods
Classic autoradiography techniques are performed according to the
following general sequential steps:
In vivo autoradiography
1. Radioactive labelling of biological sample. Labeling time depends on
the type of radioisotope and the radiotracer molecule.
• Injection or oral administration of radioactive tracer in laboratory
animals
2. Sample preparation
• Cryopreservation of euthanized animals and cryosection – whole-
body or tissue sections (20-50 µm thick) for microscopy evaluation.
Light or electron microscopy can be used, depending on the aim of the
study.
• Whole-body or tissue sections are mounted into glass slides and
embedded in photographic emulsion to generate a latent image.
3. Image development. Here, the incubation time in the developer reagent
depends only on the radioisotope used.
4. Arrest of image development by exposing the slide to a fixative reagent.

8. Whole-body autoradiography (WBA) and Quantitative Whole-body
autoradiography (QWBA) – A drug distribution in rodents.
A – intravenous administration of [14C]-ethanol (Gifford, Espaillat, & Gatley,
2008)darker areas correspond to areas of high ethanol concentration.
B – intravenous injection of [3H]-glucose (Potchoiba & Nocerini, 2004). Region of
high glucose (radioactivity) concentration are shown in green/blue.

9. Rate of DNA replication
The rate of DNA replication in a mouse cell growing in vitro was
measured by autoradiography as 33 nucleotides per second.
The rate of phage T4 DNA elongation in phage-infected E. coli was also
measured by autoradiography as 749 nucleotides per second during the
period of exponential DNA increase at 37 °C.

10. Detection of protein phosphorylation
Phosphorylation means the posttranslational addition of a phosphate
group to specific amino acids of proteins, and such modification can
lead to a drastic change in the stability or the function of a protein in
the cell.
Protein phosphorylation can be detected on an autoradiograph, after
incubating the protein in vitro with the appropriate kinase and γ-32P-
ATP.
The radiolabeled phosphate of latter is incorporated into the protein
which is isolated via SDS-PAGE and visualized on an autoradiograph
of the gel.

11. Detection of sugar movement in plant tissue
In plant physiology, autoradiography can be used to determine sugar
accumulation in leaf tissue.
Sugar accumulation, as it relates to autoradiography, can described the
phloem-loading strategy used in a plant.
For example, if sugars accumulate in the minor veins of a leaf, it is
expected that the leaves have few plasmodesmatal connections which is
indicative of apoplastic movement, or an active phloem-loading
strategy.
Sugars, such as sucrose, fructose, or mannitol, are radiolabeled with
[14C], and then absorbed into leaf tissue by simple diffusion.

12. Factors determining the 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.

13. 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.
Vector At5g23940

14. • To find and investigate the various properties of DNA
• To find the location and amount of particular substance within
a cell including cell organelle, metabolites etc.
• Tissue localization of radioactive substance.
• To find the site and performance of targeted drug.
• To locate the metabolic activity site in the cell.
Applications Of Autoradiography

15. Positron-emission tomography (PET)
It is a nuclear medicine functional imaging technique that is used to observe
metabolic processes in the body as an aid to the diagnosis of disease.
The system detects pairs of gamma rays emitted indirectly by a positron-
emitting radioligand, most commonly fluorine-18, which is introduced into
the body on a biologically active molecule called a radioactive tracer.
Different ligands are used for different imaging purposes, depending on
what the radiologist/researcher wants to detect.
Three-dimensional images of tracer concentration within the body are then
constructed by computer analysis.
In modern PET computed tomography scanners, three-dimensional imaging
is often accomplished with the aid of a computed tomography X-ray scan
performed on the patient during the same session, in the same machine.

17. Positron Isotope Half-life (min) Application
11-carbon (11C) 20.4
Detection of tumor margins and
metastasis
13-nitrogen (13N) 9.96 Myocardial perfusion imaging
15-oxygen (15O) 2.07 Hemodynamics and blood flow
18-fluor (18F) 109.7 Tumor and metastasis detection
68-gallium (68Ga) 68.3
Detection of neuroendocrine
tumors
Common positron emitting radioisotopes used in PET

18. • Single-photon emission computed tomography (SPECT, or less
commonly, SPET) is a nuclear medicine tomographic imaging technique
using gamma rays and is able to provide true 3D information.
•This information is typically presented as cross-sectional slices through
the patient, but can be freely reformatted or manipulated as required.
•The technique needs delivery of a gamma-emitting radioisotope (a
radionuclide) into the patient, normally through injection into the
bloodstream.
•On occasion, the radioisotope is a simple soluble dissolved ion, such as an
isotope of gallium(III).
•Most of the time, though, a marker radioisotope is attached to a specific
ligand to create a radioligand, whose properties bind it to certain types of
tissues.
SPECT: Single-photon emission computed tomography

20. Radioisotope Half-life Application
67-gallium (67Ga) 78 hours Bone disease
99-technecium
(99mTc)
6 hours
Bone scanMyocardial perfusion
scan
Brain scan
111-indium (111In) 2.81 days White cell scan
123-iodine (123I) 13 hours
Neuroendocrine or neurological
tumor scan
131-iodine (131I) 8.06 days
Neuroendocrine or neurological
tumor scan
Common gamma-ray emitting radioisotopes used in SPECT

21. THANK YOU