14
General Features of Radioisotopic Methodology
it as heat. This process is called chemical quenching. It results in a decrease in
general energy output of radioactive disintegration involved in the scintillation process at the solvent level. The pulse height of light emission by excited
fluor molecules consequently decreases, thus causing this apparent shift of
energy to the lower end of the chemically quenched samples. Among the especially ~trung <:hemical quenchers are gaioids, oxygen, and orgamc molecules
containing C = O-groups. Strong chemical quenching could also be induced
by acids, which contain an excess of protons. The latter intercept negatively
charged f3-particles and thus decrease the excitation sensitivity of fluor.
The colored substances often present in the biological materials, especially
those with absorption maxima close to the ultraviolet range (yellow staff),
cause severe quenching and many problems for the researcher. This kind of
quenching is called color quenching. It is specific to liquid scintillation counting of biological objects which, as a rule, contain pigments like chlorophyll
hemoglobins, cytochromes, and flavoproteins. Color quenching is a sequence
of attenuation of photons, emitted from primary or secondary fluors, by molecules of any colored impurities or by other materials which have their attenuation spectra close to the wave spectrum of the light emitted by fluors. Thus,
the molecules of colored substances in the counting sample decrease the
number of photons originally produced by the scintillation process which can
reach the phototube.
A general strategy in the practical use of liquid scintillation counting for
biological studies should be:
The selection of experimental protocols, which avoid the need to estimate the
counting efficiency K or the absolute counting rates as dpm, thus using only
relative cpm values.
The reduction of the mass of radioactive material and mineral salts per counting sample.
The appropriate solubilization of radioactive materials in the scintillation
cocktail.
The bleaching of samples with the aid of peroxides or perchloric acid.
The selection of the most appropriate and simple method of quench correction in accordance with the methodology used, and with the availability of
instrumental and reagent base.
Concerning the first point, the only stipulation is that the counting samples
should be assembled approximately equally and thus should have quasi equal
counting efficiency K. This equality must take into account the phase composition of the counting sample, the volume of cocktail in the vials, the kind and
amount of radioactive material placed in the vial, etc. Minimization of the mass
of radioactive material may be achieved by increasing its specific radioactivity in experiments. The work of researching biochemists working with tissues
and colored homogenates has been directed to developing methods providing
solubilization of various radioactive materials in the scintillation cocktails, as
this it largely improved the counting efficiency (see above).
General Features of Radioisotopic Methodology
it as heat. This process is called chemical quenching. It results in a decrease in
general energy output of radioactive disintegration involved in the scintillation process at the solvent level. The pulse height of light emission by excited
fluor molecules consequently decreases, thus causing this apparent shift of
energy to the lower end of the chemically quenched samples. Among the especially ~trung <:hemical quenchers are gaioids, oxygen, and orgamc molecules
containing C = O-groups. Strong chemical quenching could also be induced
by acids, which contain an excess of protons. The latter intercept negatively
charged f3-particles and thus decrease the excitation sensitivity of fluor.
The colored substances often present in the biological materials, especially
those with absorption maxima close to the ultraviolet range (yellow staff),
cause severe quenching and many problems for the researcher. This kind of
quenching is called color quenching. It is specific to liquid scintillation counting of biological objects which, as a rule, contain pigments like chlorophyll
hemoglobins, cytochromes, and flavoproteins. Color quenching is a sequence
of attenuation of photons, emitted from primary or secondary fluors, by molecules of any colored impurities or by other materials which have their attenuation spectra close to the wave spectrum of the light emitted by fluors. Thus,
the molecules of colored substances in the counting sample decrease the
number of photons originally produced by the scintillation process which can
reach the phototube.
A general strategy in the practical use of liquid scintillation counting for
biological studies should be:
The selection of experimental protocols, which avoid the need to estimate the
counting efficiency K or the absolute counting rates as dpm, thus using only
relative cpm values.
The reduction of the mass of radioactive material and mineral salts per counting sample.
The appropriate solubilization of radioactive materials in the scintillation
cocktail.
The bleaching of samples with the aid of peroxides or perchloric acid.
The selection of the most appropriate and simple method of quench correction in accordance with the methodology used, and with the availability of
instrumental and reagent base.
Concerning the first point, the only stipulation is that the counting samples
should be assembled approximately equally and thus should have quasi equal
counting efficiency K. This equality must take into account the phase composition of the counting sample, the volume of cocktail in the vials, the kind and
amount of radioactive material placed in the vial, etc. Minimization of the mass
of radioactive material may be achieved by increasing its specific radioactivity in experiments. The work of researching biochemists working with tissues
and colored homogenates has been directed to developing methods providing
solubilization of various radioactive materials in the scintillation cocktails, as
this it largely improved the counting efficiency (see above).