Scintillation counting chemiluminescence

After solubilisation, the sample is added to a sdntillation cocktail suitable for aqueous solutions. Solubilisation of tissues often produces chemiluminescent materials which can distort measurements of radioactivity. To reduce or remove this problem, it is recommended that samples should be kept in the dark before counting. An expensive alternative to scintillation counting for tissue samples, and one which is only justified when handling highly repetitive samples, is combustion of the sample in an appropriate furnace the 14C and 3H labelled material is converted respectively into C02 and H20 which are collected in suitable wash bottles. 32P label is converted into phosphate which remains in the ash following combustion. 

Liquid scintillation counters use two photomultiplier tubes with a coincidence circuit that prevents counting of events seen by only one of the tubes. In this way, false counts due to chemiluminescence and noise in the phototube are greatly reduced. Quenching is a problem in all liquid scintillation counters. Quenching is any process which reduces the efficiency of the scintillation counting process, where efficiency is defined as... 

INTRODUCTORY REMARKS ON PROBLEMS OF CHEMILUMINESCENCE IN LIQUID SCINTILLATION COUNTING... 

We also compared chemiluminescence and phosphorescence processes and found that the energy spectra of chemiluminescence and of phosphorescence reactions are strikingly similar. Phosphorescence reactions, however, decay completely within 20 to 30 minutes, and thus present no serious problem as a disturbing factor in liquid scintillation counting. It should be stressed, however, that samples for radioactivity measurements should be kept in the dark for about 30 minutes before the start of counting. This may be of decisive importance for scimples with... 

In the past ten years, many efforts have been made to overcome problems of chemiluminescence in liquid scintillation counting. To avoid or suppress counting errors due to chemiluminescence various chemical and physical methods as well as electronic devices have been developed and are excellently reviewed by Peng (1977). These techniques may not be effective in all cases, so it is still necessary to be aware of the possibility of interfering chemiluminescence in liquid scintillation counting. 

To quantify luciferase reporter gene expression in cell lysates, transfer 50 pi cell lysate from each well into a 96-well black flat-bottom microplate. Add 100 pi luciferase buffer per well, and optionally mix with a pipette. Measure the chemiluminescence intensity (count time 0.20 min with background correction) using a luminometer, e.g., a Microplate Scintillation Luminescence Counter (Canberra Packard) or a Wallac Victor 2 Multi-label Counter (PerkinElmer). 

Chemiluminescence (CL) was measured on all samples Immediately after plasma irradiation and before exposure to air or moisture and again after exposure to room air. CL in counts per min (CPM) was determined in a Packard 3255 liquid scintillation spectrometer equipped with low dark-noise photomultiplier tubes (RCA 4501/V4) and a Parkard 585 linear recorder. 

Fig. 32 (179). Chemiluminescence assay of bovine erythrocuprein and different Cu2+-amino acid complexes at pJi 7.8. None Cu(Lys)2 50 nM Cu(His)a, 100 nM Cu-Tyr, 145 nM bovine erythrocuprein, 8 nM. The assay components were pipetted in a disposable scintillation vial at room temperature. The total volume was 2.22 ml. The assay mixture was composed of HEPES buffer, 50 mM xanthine, 0.33 mM catalase, 800 i.U. luminol, 1 mM The reaction was started with 0.08 units xanthine oxidase (definition as given by J. Cooper, P. A. Srere, M. Tabachnick and E. Rocker, Arch. Biochem. Biophys. 74 (1958) 306). The first reading was taken after 10 sec. During the counting the coincidence of the Packard scintillation counter was turned on. The background was 4 1 cpm...

Chemiluminescence and photoluminescence are other forms of interference that can reduce the accuracy of LS techniques. Chemiluminescence describes the emission within the scintillation cocktail of photons that result from a chemical reaction common initiators are samples with an alkaline pFl or the presence of peroxides. Photoluminescence can occur when the scintillation cocktail is exposed to ultraviolet light. Some substances in the cocktail, notably the scintillator, are excited and then emit light when the species return to ground state. The effect of photoluminescence is reduced in LS systems by decay when the sample train is held in a dark environment for a few minutes prior to counting. On the other hand, chemiluminescence may have a slow decay rate that requires a change in sample preparation to eliminate the chemical that causes it. Some LS systems identify luminescence by pulse shape and indicate its relative extent. 

NotesTo avoid problems with alkaline chemiluminescence in freshly prepared counting samples it is advisable to prepare a stock solution of alkaline scintillation fluid (see conclusion )... 

FIGURE 1. Time dependent decrease of count rate of a chemiluminescence reaction measured in the H-channel and C-channel of a liquid scintillation counter. 

It is evident from Eq. (15) why it is essential for low-intensity chemiluminescent reactions to be counted in the out-of-coincidence mode for measurements made in the liquid scintillation counter. These same statistical arguments apply to fluorescence and phosphorescence measurements in the liquid scintillation counter. 

The use of the liquid scintillation counter for chemiluminescence in the out-of-coincidence mode, at phototube-amplifier gains such that single photoelectrons are detected also means that the thermionic emission of the phototube photocathode will be counted with equal efficiency. If N and N represent the thermionic electron noise of the phototubes, the signal-to-noise ratio for chemiluminescence measurements will be approximately... 

Fig. 3. Relative integral disarimination counting rate curves for phototube noise (A), for the chemiluminescence of Luminol (o) and urine ( ) and for the luminescence of a heavily quenched scintillation cocktail (A).

Since the chemiluminescence is an assay for phagocytizing cells and Eq. (56) represents a singlet oxygen generating system it is possible to use the Lumlnol chemiluminescence reaction to enhance the (Eq. (A)) and therefore to increase the sensitivity of the assay. Trush et al. (1978) report a maximum chemiluminescent intensity of 3.3 x lO count s PMN from normal PMN measured in the liquid scintillation counter. 

Alternatively, solid samples, like plant and animal tissues are first solubilized in strongly basic solutions (Hyamtne 10-X hydroxide, NCS solubilizer, Soluene, or Fh-otosol). Once the solubilization is over, scintillation cocktail is added and the sample can be counted. Here also, chemiluminescence may pose a problem. 

Chromatographic agents, solvents, and samples can frequently reduce the luminous output produced by interactions between the radioactive sample and the fluor of the scintillation fluid. This effect, known as quenching, directly reduces the counting efficiency of the system and can result in significant quantitation errors if not taken into account. In addition to quenching, other interfering processes such as chemiluminescence, phosphorescence, and efficiency losses due to self-adsorption of labeled compounds on the TLC adsorbent can affect quantitative measurement (5,49,50,51). 

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