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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Radioactive tracers
The use of radioactive tracers in cell research is an effective and safe means of monitoring molecular interactions.
There is simply no other technique which allows the precision and specificity of radioactive tracers.
Radiation is to be taken
seriously. At a minimum, its misuse can lead to increased environmental pollution, and at worst can lead to serious long term injury.
It can be handled safely, however. Radioactivity is caused by the spontaneous release of either particulate and/or electromagnetic
energy from the nucleus of an atom. Atoms are composed of a positively charge nucleus, surrounded by the negatively charged electrons.
In an uncharged atom, the number of orbital electrons equals the number of positively charged protons in the nucleus. In addition,
the nucleus contains uncharged neutrons. A proton has a mass of 1.0076 amu (Atomic Mass Units), while a neutron has a mass of 1.0089
amu.
If the mass of a helium nucleus is examined, there is a difference between the expected mass based on its proton and neutron
composition, and the actual measured mass. Helium contains two protons and two neutrons in its nucleus, and should have a corresponding
mass of 4.0330 amu. It has an actual mass, however, of 5.0028 amu. The difference (0.0302 amu) is the equivalent energy of 28.2 Mev
and is known as the binding energy . It would require 28.2 Mev to fuse two protons and two neutrons into a helium nucleus, and the
fission of the helium nucleus would yield the same energy.
In addition, the electrons orbit the nucleus with precise energy
levels. When the electrons are in their stable orbits, they are said to be in their ground state. If the electrons absorb energy (e.g.
from photons), they jump to different, yet characteristic orbits and enter the excited state. The energy difference between a ground
state and an excited state can take the form of an electromagnetic radiation.
The number of protons in the nucleus of an atom
is called the atomic number, while the number of protons plus neutrons is the mass number. The mass number is approximately equal
to the atomic weight. In the representation of an atom used in the periodic table of elements, the atomic number is a subscript written
to the left of the letter(s) designating the element, while the mass number is written as a superscript to the left.
The chemical
identity of an element is determined by the number of protons in the nucleus of the atom. The number of neutrons may vary, however.
Elements sharing the same number of protons, but having different numbers of neutrons are known as isotopes. Hydrogen, for example,
has one proton. All nuclei containing one proton are hydrogen nuclei. It may have one, two, or three neutrons. The isotopes of hydrogen
would be written as
Note that each of the three will chemically react as
hydrogen. This is important for tracer work in cell biology. The substitution of either deuterium or tritium for hydrogen in a molecule
will not effect any chemical or physiological changes in the activity of the molecule. Tritium will, however, tag the molecule by
making it radioactive.
Radiation emissions have several forms. When an atom reorganizes its sub-atomic structure to a more stable
form, it may emit neutrons, protons, electrons, and/or electromagnetic waves (energy). An alpha particle is 2 protons plus 2 neutrons
(essentially a helium nucleus). A beta particle is an electron. Gamma rays are electromagnetic energy waves similar to x-rays. The
release of sub-atomic particles and energy, resulting in the change of one element to another is known as radioactivity.
Radioactive
elements thus, by their very nature, self destruct. The loss of their sub-atomic particles is a spontaneous process, and once it has
occurred, the element is no longer radioactive. With time a percentage of all radioactive elements in a solution will decay. Statistically,
it is nearly impossible to predict which individual element will radioactively decay, but we can make a prediction about large numbers
of the elements. That is, we can say that if we wait 14,000 years, half of the radioactivity in a sample of
With a half-life of 14,000 years, radioactive carbon will be around for
a very long time. This is why it is used for dating rocks and fossils. If one makes some assumptions about the activity of the carbon
when the fossil was formed, and measures the current level, the age of the fossil may be determined.
The amount of radioactive material
is measured by how many nuclei decay each second, and this value is known as the activity. It is measured in curies Each radioisotope
has three important properties; the type of particles emitted, the particle energy, and the half-life. The energy and kind of decay
particle will determine the penetration of the radiation, and therefore determine the degree of shielding necessary to protect the
user. The half-life determines both the remaining activity after storage or use, and the time that the isotope must be stored before
disposal.
In cell biology, only a few of the many radioactive elements are used routinely. The primary elements used are
Not available at this time
TABLE H.1 Radioactive Sources and Emission Types
MEASUREMENT OF DOSE
When
alpha or beta particles, or gamma radiation pass through matter, they form ions. They accomplish this by knocking electrons from the
orbits of the molecules they pass through. We can monitor the ionization effect by allowing the radiation to pass through dry air
and measuring the numbers of ions formed. This is most often done by designing a chamber with an electrical charge capacitance, allowing
the radiation to pass through the chamber and monitoring the amount of capacitance discharge caused by the formation of ions. The
device is a Geiger-Mueller Counter and has many variations.
The ionizing ability is measured in roentgens, and a roentgen is the number
of ionizations necessary to form one electrostatic unit (e.s.u.) in 1 cc of dry air. Since the roentgen is a large unit, dosage for
cell research use are normally divided into milliroentgens (mR).
Curies measure the amount of radioactive decay, roentgens measure
the amount of radiation transmitted through matter, over distance. Neither unit is useful in determining biological effect, since
biological effect implies that the radiation is absorbed by the tissues that are irradiated.
The rad (radiation absorbed dose)
is a unit of absorbed dose and equals 100 ergs absorbed in one gram of matter. The roentgen is the amount of radiation exposure in
air, while the rad represents the amount of radiation exposure in tissue. The two are usually very close in magnitude, however, since
for most biological tissues, 1 roentgen produces 0.96 rad.
Not all radioactive emissions have the same penetrating power, however.
If radiation safety (monitoring of dose) is considered, then the rad is insufficient. A linear-energy- transfer dependent factor must
be defined for each type of emission. An alpha particle, for example, would not travel very far through tissue, but it is 10 times
more likely to be absorbed than a gamma wave of the same energy dose. This factor is known as the Quality Factor (QF) or Relative
Biological Effectiveness (RBE). The RBE is limited to work in radiobiology, the QF is used in other exposure monitor schemes. The
use of the QF results in a new parameter, the rem. The rem is a unit of dose equivalent and is equal to the product of the QF x rad.
DETECTION OF RADIOACTIVITY
IONIZATION CHAMBERS
The most common method of measuring radiation exposure is the use
of an ionization chamber. Among the more common forms of ionization chambers are the Geiger counter and the pocket dosimeter.
The
chambers are systems that comprise two electrical plates, with a potential established between them by a battery or other electrical
source. In effect, they function as capacitors. The plates are separated by an inert gas, which will prevent any current flow between
the plates. When an ionizing radiation enters the chamber, it induces the formation of an ion, which in turn is drawn to one of the
electrical plates. The negative ions are drawn the the anode (+ plate) while the positive ions are drawn to the cathode (- plate).
As the ions reach the plates, they induce an electric current to flow through the system attached to the plates. This is then expressed
as a calibrated output, either through the use of a digital or analog meter, or as a series of clicks , by conversion of the current
through a speaker.
The sensitivity of the system depends on the voltage applied between the electric plates. Since alpha particles
are significantly easier to detect than beta particles, it requires lower voltage to detect the high energy alpha particles. In addition,
alpha particles will penetrate through the metal casing of the counter tube, whereas beta particles can only pass through a quartz
window on the tube. Consequently, ionization chambers are most useful for measuring alpha emmissions. High energy beta emissions are
able to be measured if the tube is equipped with a thin quartz window and if the distance between the source of emission and the tube
is minimal.
A modification of the basic ionization chamber can be made by engineering the tube such that it is miniaturized
and such that the tube can be charged to hold a voltage without constantly rebuilding the voltage via a battery. This gives rise to
the pocket dosimeter . This device is a capacitor, which is charged by a base unit and which can then be carried as a portable unit.
They are often the size and shape of a pen and can be thus carried in the pocket of a lab coat. When exposed to an ionizing radiation
source, the capacitor discharges slightly. Over a period of time, the charge remaining on the dosimeter can be monitored and used
as a measure of radiation exposure. The dosimeters are usually inserted into a reading device which is calibrated to convert the average
exposure the dosimeter has had directly into roentgens or rems. 1 Since the instrument works by discharging the built up charge, and
the charge is upon a thin wire in the center of the dosimeter, it can be completely discharged by the flexing of that wire, as it
touches the outer shell upon impact. When later read for exposure, the investigor will be informed that they have been exposed to
dangerously high levels of radiation as there will be no charge left in the dosimeter. Besides causing great consternation with the
Radiation Safety Officer, and a good deal of paper work, it also causes some unrest with the investigator. The dosimeters should be
worn in a location where they can not impact any other objects.> Since the dosimeters normally lack the fragile and vulnerable
quartz windows of a Geiger tube, and carry lower voltage potentials, they are used for the measurement of x- ray and high energy gamma
radiation, and will not detect beta emmissions.
PHOTOGRAPHIC FILM
Low energy emissions are detected more conveniently
through the use of a film badge . This is simply a piece of photographic film sandwhiched between cardboard and made into a badge
which can be pinned or clipped onto the outer clothing of the investigator. They can be worn routinely and collected on a regular
basis for analysis.
When the film is exposed to radiation, it causes the conversion of the silver halide salts to reduced silver
(exactly as exposure of the film to light). When the film is developed, the amount of reduced silver (black) can be measured and calibrated
for average exposure to radiation. This is normally done by a lab specializing in the monitoring. Because of the simplicity of the
system, its relative low cost and its sensitivity to nearly all forms of radiation, it is the primary means of radiation exposure
monitoring of personnel.
SCINTILLATION COUNTERS
For accurate quantitative measurement of low energy beta emissions and
for rapid measurement of gamma emissions, nothing surpasses the use of scintillation counters. Since they can range from low to high
energy detection, they are also useful for the alpha emissions.
Scintillation counters are based on the use of light emitting substances,
either in solution, or within a crystal. When a scintillant is placed in solution with a radioactive source (liquid scintillation
counter), the radiation strikes the scintillant molecule, which will then fluoresce as it re-emits the energy. Thus the scintillant
gives a flash of light for each radiation particle it encounters. The counter than converts light energy (either as counts of flashes,
or as an integrated light intensity) to an electrical measure calibrated as either direct counts or counts per minute (CPM). If the
efficiency of the system is known (the % of actual radiatioactive decays that result in a collision with a scintillant), then disintegrations
per minute (DPM) can readily be calculated. DPM is an absolute value, whereas CPM is a function of the specific instrument used.
Low
energy beta emissions can be detected with efficiencies of 40% or better with the inclusion of the scintillant directly into a cocktail
solution. Alpha emissions can be detected with efficiencies in excess of 90%. Thus, with a liquid scintillation counter, very low
doses of radiation can be detected. This makes it ideal for both sensitivity of detection and for safety.
If the system is modified
such that the scintillant is a crystal placed outside of the sample chamber (vial) then the instrument becomes a gamma counter. Gamma
emissions are capable of exiting the sample vial and entering into a fluorescent crystal. The light emitted from the crystal is then
measured. Gamma counters are usually smaller than liquid scintillation counters, but are limited to use with gamma emittors. Modern
scintillation counters usually combine the functional capabilities of both liquid scintillation and direct gamma counting.
Since
all use of radioactive materials, and particularly the expensive counting devices are subject to local radiation safety regulations,
the specific details for use must be left to the institutional discretion. Under no circumstances should radioactive materials be
used without the express supervision of the radiation safety officer of the institution, following all specific institutional guidelines
and manufacturer directions for the instrument used.
RULES FOR SAFE HANDLING OF RADIOACTIVE ISOTOPES
All work with radioactive
material must be done in a tray lined with absorbant paper.
All glassware and equipment contacting radioactive material must be appropriately
labeled and kept inside the tray. The only exception is that microscope slides of labeled cells may be removed from the tray after
the drop of labeled cells has been applied to the slide and allowed to dry.
Plastic gloves should be worn when handling radioactive
material.
All waste solutions containing radioisotopes, all contaminated gloves, paper, etc., must be placed in appropriate liquid
or dry radioactive waste containers.
ALL INSTITUTIONAL REGULATIONS MUST BE FOLLOWED AT ALL TIMES.
USE OF ANY RADIOACTIVE
ELEMENT IS THE FULL RESPONSIBILITY OF THE INSTITUTION RADIATION SAFETY OFFICE AND ITS DESIGNATED OFFICERS.