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Radioisotope Techniques
Mak Oi Tong
Department of Life SciencesNational Cheng Kung University
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Radioisotope
n Atoms with unstable nuclei transform
into other stable atoms.
n Results : Release of energetic particles
or radiant energy,
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Atoms, isotopes and radioisotopes
n Atomic number, mass (mass number or atomic weight)
n Carbon : 12C6n atomic number = 6 and atomic mass = 12
n Contains 6 protons, 6 neutrons and 6 electrons.
n Isotopes : atoms have identical chemical properties butdifferent atomic weight.
n e.g. 11C, 12C, 13C and 14C
n14C6 >
+ 14N7 +e- (antineutrino)
n11C6 >
+ + 11B5 +e (neutrino)
n Ratio of neutrons and protons
n Lighter atoms 1:1 and 1.5:1 at high atomic number
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Types of radioactive decay
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Negative-Beta (electron) emission
n Ejected electrons emit from the nucleus
which are identical with the orbital electron in
mass and charge (neutron to proton).n
3H1 >3He2 +
+e- (antineutrino)
n14C6 >
14N7 + +e- (antineutrino)
n
32
P15 >32
S16 +
+e- (antineutrino)n
35S16 >35Cl17 +
+e- (antineutrino)
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Positive-Beta (positron,+) emission
n + is same mass as electron but having opposite
charge, and it will emit as gamma (
) radiation.n + + e- > (1.02 MeV)
n11C6 >
11B5 ++ +e (neutrino)
n13N7 >
13C6 ++ +e (neutrino)
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Decay by electron capture
n Conversion of a nuclear proton into a neutron
by capture one of the two electrons in theinnermost K-shell, ie. K-capture (KC) and
emission of a positron+.
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Decay by-radiation
n -Ray emission occurs after or+
emission.
D b i l i i
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Decay by- particle emission
n Heavy atoms with more than 82 protons will
emit an-particle which is a helium nucleus
of atomic mass number of 4 (2 protons and 2
neutrons) with a charge of +2.
n226Ra88,
235U92 etc. are examples for-
particle emitters and are not for biochemical
studies but for radiological toxicity.
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radioactive isotopes
n Pierre and Marie Curie introduced the term
radioactivity to designate the process of spontaneousemission of radioisotope.
n Artificial production of radioisotopes is normallydone by neutron bombardment.
n Other methods: -particle, proton and deuterons
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n To-day, over 1500 isotopes are producedartificial produced.
n On earth, radioisotopes are produced byneutral bombardment, and 14C and 3H(tritium) are in equilibrium.
n Radioisotopes can be used for radiocarbon(14C) dating.
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Properties of radioactive emissions
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Energy of-particle emissions
n -
,+
and e-
travel near the speed of light, 3x 1010 cm/s or 3 x 108 m/s.
n Emax is the maximum energy value, and the
Eave Average) is equal to 1/3 of Emax.
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Interaction of particular with their
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Interaction of -particular with their
environment
n - will cause electron excitation and emit UV or
visible light.n In air, about 34 ev is required to produce one ion
pair.
n An ejected electron, with an energy of 1.5 MeV,will travel about 2 meters in air, and about 1 cm intissue.
n Eventually the ejected electron will be captured by
an empty electron orbit of a positive ion.n When a positron encounters an its electron, the two
particles annihilate (combine) each other and theirmasses are converted into electromagnetic-
radiation which is I the form of two 0.51-Mev
amma ra at on an ts nteract on
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amma ra at on an ts nteract onwith matter
n Gamma rays are high energy photons or light
particles that are electrically neutral with zero massand consequently can penetrate matter readily.
n -Ray is emitted from the nucleus with discreteenergy range between 10 KeV to 3 MeV.
n Many isotopes emit of- ray of several energies indiscrete steps (Fig. 6.2).
n Photoelectric effect is a- ray of relatively low
energy (
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n The Compton effect results when- rays of
medium energy (>0.1 MeV) undergo elastic
collisions with loosely bound orbital electron and aportion of the- ray energy is transferred to the
ejected electron.
n
The photon is deflected in a new direction withreduced energy and undergo several more similar
collisions before losing all its energy.
n Compton electrons have a wide range of energies
and these electrons will produce secondaryionization as they travel through the medium,
dissipating their energy.
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n Pair production results when a relativelyenergetic (>1.02 MeV)- ray interacts with
a nuclear force field.n The photon has all its energy converted into a
positron and an electron.
n This is essentially the conversion of energyinto mass.
n Both the ejected positron and electron may
cause ionization of atoms in their paths, andcollies together.
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Kinetics of radioactive decay
n The process of decay of a radioactive nucleus into a
stable, nonradioactive nucleus is irreversible.
n Rate of decay (particle emission) is equal to the rate
of disintegraton of the nuclei, which is proportional
to the number of radioactive nuclei present.
n Rate of decay decreases exponentially with time.
n It is independent of pressure, temperature and other
chemical and physical changes.n The rate is first order reaction.
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n Rate of decay:
n N = amount of radioactive nuclei present
n = decay constant
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n Activity is the quantitative measurement of
radioactive and if the activity of sample is A
(by detector), it becomes the counting rate ofthe sample.
Efficient of detector
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Half-life (t )
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Units used in radioactive measurements
n The electron volt
n One electron volt (1 eV) is the kinetic energydeveloped by an electron when it is acceleratedthrough a potential difference of 1 volt in a
vacuum.n 1 eV is equal to 3.85 x 10-20 calories or 1.602 x
10-19joule.
n It is normally expressed in KeV or MeV.
n The electron emitted from 14C has an averageenergy of 4500 eV per nucleus (atom).
Th i h i f di i di i i
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n The curie: the unit of radioactive disintegration
n One curie is equal to the number of disintegration per
second in 1 gram of pure radium 226.
n It is also equal to 2.22 x 1012 disintegration per minute
(dpm).
n One becquerel (one disintegration per second) is equal to
3.70 x 1010 becquerels.
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Safety in handling radioisotopes
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Safety in handling radioisotopes
n First to know whether it is- or+ -ray isotopes.
n Prevent injection, inhalation or absorption ofradioisotopes in human body.
n Laboratory coat, plastic gloves etc. Should be worn for
body protection.n Restricted areas for handling radioisotopes must be
labeled, clean and neat.
n Covered with plastic absorbent paper with a tray line.
n Have special containers for any radioisotope disposal
such as glasses, plastic, paper, water and organic
solvent.
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n Monitor the working place routinely for any
contamination of radioactivity by using Geiger-
Muler survey monitor for strong and ray.
n Wear a personal dosimeter for high energy
radioisotopes.
n Wash immediately if any contamination is present.n If skin has contacted with radioisotope, wash with
immediately for at least 2 minutes.
n Special care is required for anyone handling 125I or131I because of the effects on thyroid gland in
human body.
Statistics of counting radioisotopes
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Statistics of counting radioisotopes
n Compare Fig. 6.1 and Fig. 6.5.
n Standard deviation s:
n A more common situation is:
n Confidence of the true value.
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P l i d d d i i (% RSD)
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n Percent relative standard deviation (% RSD)
n For sample comparison, % RSD is generally
required.
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Measurement of radioactivity
n By gas ionization.
n By scintillation counting
By gas ionization
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By gas ionization
n When a energetically charged particle, such
as- particle, passes through a gas, itselectrostatic field displaces orbital electron
fro the gas molecules close to its path.
n This displacement produce a positive ionfrom the other part of the molecule, resulting
an electron pair.
By gas ionization
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y ga a
n If an electric field is applied in the chamber,
the displaced electrons are accelerated towardthe anode and the positive ions move towardcathode slowly (because of their larger mass).
n
If higher electric fields are applied, theelectron displaced by the first- particlecollision is accelerated and collides with othergas molecules to produce additional ion pairs.
n This result is an amplification of the numberof electrons from the first electron, giving ashower of electrons.
monitor
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n A type of radioactive detector.
n It is a gas-filled tube with cylindrical cathode
and a fine wire anode.
n A common gas mixture is the so called Q-gas
which is 1.3 % isobutane in helium.
n One end of the tube has a window through
which the- particles from the radioactive
sample.
monitor
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n A voltage potential is applied between the electrodes
of the tube and produces shower
n The electrons are collected at the anode within amicrosecond, and a strong electrical pulse is formed.
n The number of electrons collected is proportional to
the number of initial ionization, and is proportionalto the number of- particles entering the chamber.
n The region is called proportional region.
n
Efficient, for
14
C about 1 to 5 % and for
3
H is nearlyzero.
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By scintillation counting
n Use scintillator or fluors for- and- ray counting.
n Scintillator can absorb radiant energy to form excited atoms
or molecules and return rapidly to ground state, releasing
excitation energy, to form photon and heat.
n Amount of photon released directly related to the amount of
radiant energy.
n
Liquid scintillation counting is that the scintillators aredissolved in a solvent called cocktail.
By scintillation counting
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y g
n Steps for photon formation:
n - Radiant energy is absorbed by solvent and become excited.
n The solvent transfers the the excited energy to the scintillator to
cause excitation.
n The scintillators release photon which is detected by the phototubes.
n The phototube converts photons into amplified numbers of electron,and then becomes voltage pulse by a pulse height analyzer, and this
is called- and- scintillation counter.
n - energy spectrum refers to Fig. 6.12.
n
Dual-labeled mixture activity determination refers to Fig.6.13.
Cocktail composition and samplepreparation
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preparationn Primary fluor is 2,5-diphenyloxazole (PPO).
n Secondary fluor is 1,4-bis-2-(5-phenyloxazolyl) benzene(POPOP).
n Common solvents: toluene and dioxanen Toluene is used for organic compounds.
n
Dioxane is used for aqueous samples.n Heterogeneous counting is that the radioactive samples are
in solid support.
n Homogeneous counting is that the radioactive samples arein dissolved form.
n Background counting is caused by electronic noise, cosmicradiation and other natural radioisotopes present.
n Cerenkov counting is used for direct- particle countingwithout fluor, only for high energy- emittor, e.g. 32P.
Quenching
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n It is the process that causes a reduction in the amount of
fluorescence produced by
- emission in a liquidscintillation cocktail, reduction amount of light reaching the
photomultipler tube.
n Chemical quenching is that the transfer of energy is
interfered from solvent to fluor molecules (fluorescencereduction).
n By acid
n By dipole-dipole interaction of quenching agent
n Electron capture by quenching agent.
n Color quenching is the presence of colored compounds
which can absorb the photons emitted by the fluor, e.g.
Pigment, blood.
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Quench correctionn
Internal standard method:n Add another non quenching radioactive standard and
recounted.
n External standard method:
n Use of a highly radioactive- radiation source, e.g.226Ra or 137Cs, which is brought near to the test
sample vial and the sample is recounted in a short
time.
n Efficient by ES refers to 6-19.
S lid i till ti ti f di ti
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Solid scintillation counting of radiation
n
A crystalline inorganic fluor, sodium iodide (NaI), withsmall amount of thallium (Tl) as activator.
n - Rays interact with crystalline fluor to form ion pair, the
Compton effect and induce photoelectric and excite
crystalline fluor, finally produce photon to reachphotomultiplier tube.
n The crystalline fluor is sealed in a metal cylinder from
external light.
n The cylinder must be hermetically sealed because ofhygroscopic of NaI.
n It is designed for the counting of125I, 51Cr and 60Co.
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A toradiograph
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Autoradiography
n A technique for the determination the location of
radioisotopes in tissue, tissue sections, chromatograms and
gels.
n It is a non-destructive method for radioactive compounds,
and can be isolated and recounted.
n Results are shown on a X-ray film and developed.
n The- ray hits the silver bromide of the film and reducing
to metallic silver from silver ions, and black spots will be
appeared.n For best darkening of the film, at least 1 x 106 (- particle)
passing through the film per cm2 per minute.
A di h
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Autoradiography
n If sample contains 1 x 103 cpm/cm2, it will take about 100
minutes or 17 hours to induce the black spot.
n For weak ray, enhancer ( fluorography, PPO) is need to
enhance the sensitivity, particularly 3H.
n The photoelectric effect are more stable under -50 to -80C
about 4x more stable.
n The resolution can be increased by decreasing the thicknes
of the sample and the distance between sample and the fil
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M h d f l b li bi h i l
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Methods of labeling biochemicalcompounds
n Two general methods:
n biosynthesis and
n chemical synthesis.
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Biosynthesis
n Used by either whole organisms (cells) or specific enzymesto synthesize radioactive compounds.
n The common used radioisotopes are 14C, 32P and 35S.
n Factors for the successful use of biosynthesis:
n By using whole organism, labeled compound(s) must beaccumulated in large number from starting compound.
n For using a specific enzyme, the enzyme must be easily isolatedand the substrate is readily available, and the labeled compoundmust be isolated and purified from the reaction mixtures.
n
For using microorganism, labeled compound is isolated from themicroorganism or the cell-free culture supernatant fluid.
n Whole organism biosynthesis gives a relatively largenumber of labeled products but enzyme synthesis gives onlyone or specific labeled products.
Ch i l h i
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Chemical synthesisn
Products can be labeled in one or few positions.n Starting materials always be scarce and expensive.
n Results give low yield.
n Give a racemic mixture of optical isomers, e.g. D- and L-
isomers of amino acids.n Types of labeling of compounds:
n Labeling of proteins.
n Labeling of nucleic acids.
n Formation of14C carbon-carbon bonds.
n Synthesis of tritium labeling [3H] compounds from NaB3H4.
n Synthesis of35S-labeled L-cysteine
T f di h i l l b li
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Types of radiochemical labelingn
Isotopically: one or more radioactive atoms have replacedstable atoms of the same element, e.g.
n3H1
1H1,14C6
12C6,35S15
31P15
n They are the same molecules.
n
Nonisotopically: a radioactive isotopes has beensubstituted for a stable atom of another element, e.g.
n125I for 1H1
n Three forms of labeling positions:
n
Specific: [1-14
C]-D-glucosen random: randomly distributed (G-).
n *CH3-CH2COOH, or CH3-*CH2COOH or CH3-CH2*COOH
n Uniform: all related atoms are labeled, [U-], e.g. [U-14C]-
sucrose.
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Radioimmunoassayn Ab (antibody) + Ag (antigen) Ab-Ag complex
n Present an abstract about the basic principle and
application of radioimmunoassay.
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Designing a radioisotope experiment
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Designing a radioisotope experiment
n
First and foremost in the design of anyexperiment is the formulation of the question tobe studied, the purpose and nature of theexperiment.
n Next is what experimental techniques toanswering the question.
n Is radioisotope necessary ? What type ofradioisotope ?
n
Which position and and which type of labeledcompound.
n Availability of the labeled compound.
n How much specific activity and amount of
radioactivity required
Designing a radioisotope experiment
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Designing a radioisotope experiment
n What is the cost of the labeled compound ?n Experimental methods.
n What type of detection system and its efficiency ?
n The dilution effects and the yield of theradioisotope.
n Control and correction factors needed.
n
Expression and interpretation of the results, andfinally
n The possible isotope effects and possible effects
of radiation damage.