SPA - General Scintillation Counting , Instrumentation and Color Quench

Q. How is radioactivity converted to light?
Q. How is light detected from a radioactive sample?
Q. How does a photomultiplier tube or PMT work?
Q. What is the difference between the TopCount and a Microbeta2?
Q. How long should I count a plate for?
Q. What is the difference in SPA counting efficiency as compared to liquid scintillation counting?
Q. What is the relationship between CPM, DPM and Counting Efficiency?
Q. How is the counting efficiency determined on the TopCount or MicroBeta2?
Q. How is the QIP value ( tSIS or SQP(I) ) related to counting efficiency?
Q. Can anything interfere in the process of light emission from a sample?
Q. What is quenching?
Q. Is all quenching the same?
Q. Is chemical quenching a problem in radioactive or SPA measurement?
Q. What is a pulse height spectrum?
Q. What is Calibration, Normalization and Standardization?
Q. How can I tell if all of my PMT's are functioning the same?
Q. What is SPQ(I) or tSIS?
Q. What happens to a pulse height spectrum or energy curve when it is quenched or what is sample quenching?
Q. What effect will colored compounds have on my sample?
Q. How can I correct for the influence of colored compounds that I may have in my samples?
Q. What are some of the general guidelines that I should follow when setting up a quench curve?
Q. What should a typical quench curve look like?
Q. Can I modify or edit a quench curve?
Q. Can I take the numbers from one quench curve and install on another counter?
Q. Why is Tartrazine used to make quench curves?
Q. Should I construct a different quench curve for every possible color I may have in my compound library?
Q. What is the best to use, total counts or labeled beads for quench curves?
Q. Does PerkinElmer have a 33P or 14C quench curve kits?
Q. How do I set up my instrument for reading a quench curve?
Q. What does 'Standard Set DPM or Isotope Activity' mean?
Q. Under what conditions do I count my quench curve?
Q. How accurate are quench curves at low count conditions?
Q. Can I place the quench curve anywhere on the plate I want to?
Q. What kind of plates do I have to use with my SPA assays and quench curves?
Q. What effect does concentrating the beads on my assay?
Q. What isotopes can cause non-proximity effect or NPE?
Q. What count mode should I be using for either YSi or PVT SPA beads?
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Q. How is radioactivity converted to light?
A. When an isotope decays, it gives off a beta or gamma particle. In quantifying radioactivity, the energy from these particles can interact with either a liquid or solid scintillant. A scintillant contains solvent molecules along with primary and sometimes secondary fluor molecules. (Since most assay development and screening labs use beta counters to quantify radioactivity, that is the type of counting will focus on.) When a beta particle or electron interacts with a solvent molecule it transfers its energy to that solvent molecule. That energy is again transferred to flour molecules, raising those molecules to an activated state. When these fluor molecules decay and return to their ground state, the energy is released in the form of photons or light, which is at particular wavelength and characteristic for that type of scintillant.

Q. How is light detected from a radioactive sample?
A. Light is detected by use of a scintillant, which gives off packets of photons or light, which is then quantitated by a device called a scintillation counter. Light output is detected by a photomultiplier tube or PMT and converted into electrons. Within the instrument, these electrons are then characterized and quantitated.

Q. How does a photomultiplier tube or PMT work?
A. A photomultiplier tube is a device that can detect low levels of light or photons at a particular wavelength. Photons that strike a photocathode are turned into secondary photoelectrons. These photoelectrons are amplified by dynodes to become secondary electrons, which are converted into an output pulse. As with all electrical energy, these secondary electrons are characterized by both number and intensity or associated energy. Thus the output pulse from a sample being measured is directly proportional to the input of intensity and frequency (number up pulses) from that original signal. The output pulse is further characterized by a multi-channel analyzer and associated circuitry.

Q. What is the difference between the TopCount and a Microbeta2?
A. To answer this question, we must focus on the theory of operation for these two instruments and associated liquid scintintillation counting. Therefore, we will only focus on the different methods of counting.

The TopCount uses unique and patented time-resolved circuitry using one PMT while the Microbeta2 uses two PMT's with coincidence circuitry. This type of circuitry is found in all other types of liquid scintillation counters, LSC's.

Microplate counters such as the Microbeta2 use coincidence circuitry, therefore they have two functional PMT's that read both the top and bottom of a sample. Each PMT must detect a signal (light output) from a single isotopic decay event within the coincidence resolving time, hence coincidence circuitry. This time is generally around 20 nanoseconds. Most isotopic decay events occur within five nanoseconds. So any event that occurs outside of a designated 20 nanosecond window is rejected as noise. Background noise is normally characterized by a single event but with less associated energy as compared to a true decay event. Also, noise is not usually seen in both PMT's at the same time. Therefore, due to coincidence circuitry, noise is ignored and not counted. But if both PMT's detect a signal within the coincidence resolving time, it is counted as a single event. To ensure that a true decay event has enough energy associated with it and therefore detected, fast scintillators are commonly used. Fast scintillators are normally used for instruments that employ coincidence circuitry. Fast scintillators collect most of the energy given off by a single decay event and emit it as one highly energetic discreet pulse.

In contrast, slow scintillators are normally used for time resolved circuitry, as found in the TopCount, which uses one PMT reading from the top of the sample. Slow scintillators allow for the energy associated with a decay event to be liberated at a lower energy level. Also, this energy is spread out over a longer period of time (about 200 nanoseconds) in the form of after pulses. As stated previously, background noise is characterized by one discreet pulse. These attributes allow for the discrimination between noise and actual decay events by TopCount's time resolved circuitry. Depending on the type of scintillator or SPA bead used, either one or two after pulses will be given off for a total of two or three pulses, respectively. Comparatively, PVT beads give off two pulses while YSi beads give off three pulses.

As implied, slow scintillators can be used with coincidence circuitry but fast scintillators can not be used with time resolved circuitry.

Q. How long should I count a plate for?
A. In either attaining DPM or CPM data, one should count a plate long enough to obtain at least 3000 total counts per sample. This will ensure that enough statically precise data will be accumulated for the formation of an accurate pulse height spectrum from which other extrapolations are made.

Q. What is the difference in SPA counting efficiency as compared to liquid scintillation counting?
A. SPA counting is not as efficient as liquid scintillation counting. Typically, counting efficiency of PVT SPA beads will be 40% and YSi SPA beads will be 60% of the expected liquid scintillation counts.

Q. What is the relationship between CPM, DPM and Counting Efficiency?
A. CPM stands for counts per minute. DPM stands for disintegration's per minute and counting efficiency refers to the ratio between the number of measured counts (CPM) and decays per unit time, usually in minutes (DPM). This can be seen by the equation: CPM = DPM x counting efficiency. Or DPM = CPM/counting efficiency or percent counting efficiency = CPM/DPM x 100.

Q. How is the counting efficiency determined on the TopCount or MicroBeta2?
A. Counting efficiency is related to the ability of either instrument, the TopCount or MicrBeta, to detect or count a sample thus obtaining CPM and a quench index parameter (QIP) number which is then related to a quench curve. Counting efficiency is extrapolated from a quench curve that is setup and installed for each assay run if DPM are required. DPM is required under most, but not all, screening conditions to correct for possible quenching of assay samples by various colored compounds. The highest point on the quench curve, is represented by the unquenched "positive standard" and is correlated to the number of CPM counts inputted as being either the Standard set DPM or the Isotope activity, depending on the instrument. This sample and the CPM value inputted for it is considered to be have a counting efficiency of 100% or 1as long as the value inputted equals the unquenched value in your quench curve. This value is correlated to a numerical level of quench or a quench index parameter, being either tSIS or SQP(I) when the quench curve is constructed. Again, this should be the highest value on your quench curve. The next point on your quench curve may represent a sample as being ~5% quenched. This will have a lower QIP value and a lower counting efficiency. Twelve samples are usually assayed in order to set up a quench curve which ranges from zero quench to 95% quench of your assay total CPM. A curve is then constructed with the QIP values plotted on the X-axis and the corresponding counting efficiency on the Y-axis. This graph is stored in the counter and used to obtain percent counting efficiency which equals CPM/DPM x 100. Once a quench curve is counted and installed, the instrument will try to correct for any quenching that may have occur in the sample when DPM is selected for data output. If there is no quenching and the counting efficiency is 100% or 1 then according to the equations above, CPM will equal DPM and no correction is made.

Q. How is the QIP value ( tSIS or SQP(I) ) related to counting efficiency?
A. If the QIP value ( tSIS or SQP(I) ) decreases, this means that there is an increased amount of quench occurring in the sample. This will reduce the counting efficiency.

Q. Can anything interfere in the process of light emission from a sample?
A. Yes, if there is any decrease in light emission from a particular sample, it is called quenching.

Q. What is quenching?
A. Quenching is a phenomenon that leads to a decrease in energy emission or light output from a particular sample that is being quantitated. This phenomenon is not confined to radioactive measurement but is applicable to any type of system that measures energy or light output from a particular sample. Other technologies that can affecting by quenching are fluorescence, chemi-luminescence, and bio-luminescence.

Q. Is all quenching the same?
A. No. There are three main types of quenching associated with radioactivity. They are color, chemical and physical quenching. Physical quenching or optical quenching involves the physical blockage of energy emission or light output from a sample. An example would be if some type of flocculent material is in the sample thus decreasing the available light reaching the detectors. This is usually not a problem as the interfering material can be easily removed. Chemical quenching is dependent on the type of scintillant being used. Any chemical that contains ringed structures such benzene and its derivatives and/or contains many multiple carbon-carbon bonds may absorb the energy from the beta particle directly or from the solvent or fluor molecules contained within the scintillant. Chemical quenching is concentration dependent and usually occurs before light emission from the fluor molecules. Color quenching is similar to chemical quenching. Like chemical quenching, color quenching is dependent upon the size and structure of the molecule. Many large molecules can absorb light at a particular wavelength, hence having a characteristic color associated with them. These molecules are often called dyes. Various dyes either; red, blue, green, or yellow in color absorb light at defined wavelengths. So any dye or colored compound that is added to a sample being quantitated, has the potential to absorb the light being emitted from that sample. As implied, color quenching occurs after light is emitted from the fluor molecules. The end result would be a decrease in the amount and intensity of light or signal being emitted.

Q. Is chemical quenching a problem in radioactive or SPA measurement?
A. Usually it is not but it depends on many factors associated with the sample or assay being measured. Chemical quenching is dependent upon the type of chemical and its concentration in the sample. Usually these chemicals are some types of solvent used to keep a compound in solution for screening purposes. At those typical concentrations chemical quenching is usually not a problem in SPA. Additionally, for a solvent or chemical to decrease light from a sample, it must interfere with the transfer of the energy from the beta particle to the scintillant. This is not possible if one is using the glass YSi beads. The chemical can not enter the glass matrix and thus interfere with the energy transfer process. Similarly, the concentration of solvents typically used in screening do not effect the plastic PVT SPA beads. But if the concentration of a particular solvent is high enough, then it may partially dissolve the PVT bead thus interfering in the transfer of energy to the scintillant and subsequent light output from the bead. These high concentrations of solvent are seldom and rarely used in a screening environment. So chemical quenching is not a problem with YSI beads and rarely seen with PVT beads.

Q. What is a pulse height spectrum?
A. A pulse height spectrum is a representation of the average kinetic energy associated with the decay of a particular isotope. When an isotope decays it liberates an electron or beta particle and a neutrino. The energy associated with this decay is randomly distributed between the two particles. The neutrino is not detected but the beta particle can be detected by a PMT as light energy. This is due to the interaction of the beta particle with either a liquid or solid scintillant molecule. The amount of light energy given off is proportional to the amount of energy associated with the beta particle. That energy is plotted on an energy axis ranging from 0.0 to Emax, a specified endpoint as determined by the instrument manufacturer. This energy is captured and correlated to defined window settings in the instrument. This energy axis is usually the X-axis while the Y-axis represents the number of counts which, is dependent on the specific activity of that isotope and or the overall count time. The result is an accumulation of counts that form an energy curve representative for that isotope. A pulse height spectrum can be used to determine if a comparative sample is being quenched.

Q. What is Calibration, Normalization and Standardization?
A. Calibration is the adjustment of the gain on the photomultiplier tubes to give the same pulse heights [SQP(I) and tSIS] values for all detectors. This is usually done by the instrument service personal. Normalization is the calculation of mathematical factors used to give the same CPM values or each photomultipler tube. Standardization is the construction of quench curves and calculation of corrected CPM values or DPM values based on counting efficiencies.

Q. How can I tell if all of my PMT's are functioning the same?
A. To determine if all the PMT's are performing adequately, one should dispense an equal amount of radioactivity into all wells of a 96 well microtiter plate. The plate should be counted and both CPM and the QIP values, either tSIS or SQP(I) obtained. Both of these values should have a 5% CV or less. If a problem is suspected, the plate should be flipped around and counting in a different orientation and the values compared accordingly. If a problem is noticed, then the appropriate service engineer should be contacted.

Q. What is SPQ(I) or tSIS?
A. For each sample counted a pulse height spectrum is generated. This spectrum is used to calculate a specific number representative of the average energy or the intensity of the light output for that isotope as detected by the PMT's. For the Microbeta2, this value is called the spectral quench parameter of the isotope or SQP(I). For the TopCount, this value is called the transformed spectral index of the sample or tSIS. Both of these values are referred too as quench index parameters (QIP's) and are mathematically determined by the instrument for every sample counted. Each instrument manufacturer has their own method of determining QIP values. These values are used to determine if a sample is being quenched as compared to a known sample with no known quenching.

Q. What happens to a pulse height spectrum or energy curve when it is quenched or what is sample quenching?
A. When the energy represented in a pulse height spectrum for a particular isotope is quenched or decreased, the energy curve shifts to the left. This decrease in energy can be quantitated by a number called the quench index parameter or QIP. For the Microbeta2, their QIP value is called the spectral quench parameter of the isotope or SQP(I). For the Packard TopCount, their QIP value is called the transformed spectral index of the sample or tSIS. As implied, when a sample is quenched, the QIP value decreases as compared to a sample that exhibits no quenching. This is indicated by decrease in CPM and counting efficiency and corrected for by DPM measurements.

Q. What effect will colored compounds have on my sample?
A. It depends on the color of the compound. Colored compounds frequently decrease the amount of light that will reach the PMT and thus detected and quantitated. Both types of SPA beads emit light at around 400nm. Any colored compounds that absorb light at or near this wavelength will have the greatest ability to decrease light output from the sample and thus quench that particular sample.

Q. How can I correct for the influence of colored compounds that I may have in my samples?
A. To correct for the presence or probable presence of colored compounds in liquid scintillation counting, one must construct and install a quench curve for that particular instrument. The data from one quench curve can not be installed on another counter. Each counter must count its own quench curve as not all PMT's function the same. If needed, quench curves can be edited. Most liquid scintillation counters allow the user to edit quench curves.

Q. What are some of the general guidelines that I should follow when setting up a quench curve?
A. We recommend that you use the same assay conditions that you are going to be screening at. This includes same weight of beads, same buffer and components, same assay volume, same count conditions, and a suitable figure for your 'Standard set DPM' or 'Isotope Activity'. Preferably identical to your assay total.

Q. What should a typical quench curve look like?
A. The most important feature of any quench curve whether it was generated on the TopCount or the Microbeta2 is that it be as smooth as possible. Any portion of the curve that is uneven can lead to large discrepancies in counting efficiency and DPM.

Q. Can I modify or edit a quench curve?
A. Yes. Most liquid scintillation counters including both microplate counters, the TopCount and the Microbeta2 allow the user to modify their quench curves.

Q. Can I take the numbers from one quench curve and install on another counter?
A. No. The data from one quench curve can not be installed on another counter. Each counter must count its own quench curve as not all PMT's perform at the same level. The performance of the PMT can effect overall counting efficiency.

Q. Why is Tartrazine used to make quench curves?
A. Both SPA beads, PVT and YSi, emit light or photons around 400nm which is the same wavelength that corresponds to the color yellow. Therefore, any yellow colored compound, such as Tartrazine will have the greatest ability to absorb light that is emitted at this wavelength thus quenching light output.

Q. Should I construct a different quench curve for every possible color I may have in my compound library?
A. Yellow, orange, and red colored compounds have the greatest overlap of emission spectra of SPA beads, which is around 400nm. Other colored compounds often have absorption spectra that overlap with the emission spectra of SPA beads but often to a lesser extent. Therefore, these compounds will have some ability to quench light output from SPA beads. To this end, to quench light emission by SPA beads one would need a certain amount of a yellow compound or dye such as Tartrazine. If one wanted to use another colored compound or dye to construct a quench curve, it is possible, but more of that compound would have to be used to produce the same quenching effects as that of Tartrazine.

Q. What is the best to use, total counts or labeled beads for quench curves?
A. Using labeled beads are the preferred method of developing a quench curve. Labeled beads are much easier to handle and incorporate into your screening chemistry that setting up your assay total for all values used in your quench curve. This leads to less variability in your data and higher precision in obtaining your quench curve.

Q. Does PerkinElmer have a 33P or 14C quench curve kits?
A. No, we do not have a 33P or 14C quench correction kit. We advise you to use your 'positive assay total' or assay conditions which give you your total CPM's to generate a quench curve. You would follow the same direction as those found in the quench correction kits but you would need to vary the amount of Tartrazine so that you cover the range of quench that may be observed in your screening environment, 5% to 95% quench of your assay totals.

Q. How do I set up my instrument for reading a quench curve?
A. Setting up your instrument, either the TopCount or the Microbeta2 means that you will be requesting your data output in terms of DPM. To this end, see the section on constructing and installing quench curves. (from the electronic version of making your quench curves)

Q. What does 'Standard Set DPM or Isotope Activity' mean?
A. Standard Set DPM is used for the TopCount and Isotope Activity is used for the MicroBeta2. This is a number that represents the maximal amount of counts (CPM) in your assay which will be entered into the instrument programming and used to determine counting efficiency.

Q. Under what conditions do I count my quench curve?
A. You count your quench curve under the same equilibrium conditions at which you will be screening your assay. The main types of bead counting conditions are floated, (PVT beads only) suspension (PVT beads only) and settled or centrifuged.

Q. How accurate are quench curves at low count conditions?
A. To generate a statistically accurate pulse height spectrum for a sample enough counts have to be accumulated. To achieve this, 3000-5,000 total counts need to be accumulated per sample. If any sample whose counts falls below this point, it is recommended that count condition be amended to accumulate enough counts to achieve an accurate pulse height spectrum. It is from this spectrum that a quench index parameter (QIP) and counting efficiency are determined. Hence, any deviation in the pulse height spectrum can result in a change in QIP, counting efficiency and hence DPM inaccuracy.

Q. Can I place the quench curve anywhere on the plate I want to?
A. It depends on the type of microplate counter you have. The TopCount allows you to put the quench curve anywhere on the plate and in any number of replicates. But it does not normalize when you do a quench curve. Conversely, the Microbeta2 will normalize when you set up a quench curve but you must put the quench curve in a defined order and only use single samples.

Q. What kind of plates do I have to use with my SPA assays and quench curves?
A. Most any type of plate can be used for SPA. Microplates that have high reflective optics associated with them are preferable to use. Additionally, Corning Costar makes a microplate (NSB Plate) specifically for use with homogenous assays. This plate is designed to reduce the non-specific binding of radio-labeled ligand to plate walls that could cause an increase in your background signal. They may not be useful for all applications but should be considered as a tool that may reduce the non-specific binding of your labeled compound to the microplate.

Q. What effect does concentrating the beads on my assay?
A. When a radiolabeled ligand is attached to the SPA bead via a capture molecule there is a 50% chance that the released beta particle or electron, will strike the bead and a 50% chance that it will travel away for the bead. This is called 2  counting. When an isotope is completely surrounded by scintillant, the released particle can be captured and detected in any geometry. This is called 4  counting and is twice as efficient at 2  counting. 4  counting can be approximated with SPA beads by concentrating the beads by one of three mechanisms, either flotation (3M CsCl2 final in well concentration), centrifugation or settling. The higher the isotope energy, the greater the difference that can be seen between 2  and 4  counting efficiency. So concentrating the beads in a 33P kinase assay will have a greater effect on counting efficiency and hence relative CPM than a tritiated ligands. Also, concentrating the beads will help reduce your non-specific background of which non-proximity effect or NPE is a component. The principle here is to reduce the proximity of SPA beads from unbound high energy isotopes. These isotopes emit a beta electron that can travel long distances in solutions, which have the ability to interact with SPA beads of which they are not specifically bound too. If this occurs, it is called non-proximity effect or NPE. This is also covered in the kinase section.

Q. What isotopes can cause non-proximity effect or NPE?
A. Any isotope can cause NPE but is more pronounced with high energy isotopes such as 35-Sulfur and 33-Phosphorus. As a general rule and if possible, all SPA assays should be counted under equilibrium conditions employing some mechanism of bead concentration. Tritiated compounds are least effected by NPE and therefore could be counted while in suspension.

Q. What count mode should I be using for either YSi or PVT SPA beads?


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