LIQUID SCINTILLATION COUNTERS. {Beta Counters}

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1 LIQUID SCINTILLATION COUNTERS {Beta Counters} We offer a range of Alpha, Beta & Gama counters, from Hidex Oy, Finland to meet your specific Liquid Scintillation counting requirements. Triathler LSC Sense LSC Plate Reader 300 SL TDCR LSC Can count samples from 200 uls - 20 ml 96 / 384 well plate 4 / 5 ml or 20 ml vials Research & Educational Research & Educational Pharmaceutical Industry Institutions Institutions & CRO s When the sample no. are less For screening of NCEs, ADME Tox More useful when using Receptor binding & Cell Toxicology studies Native tissues Proliferation assays. Mandatory for radioactive contamination monitoring specially when using 3H Economical, costs appr.. half High through put Quench Correction by or one third the price of a Conventional Multi Vial LSC TDCR Portable : Can be shifted from Lab to Lab Latest design & technology 1) Why are Multi Vial LSCs mostly used in Research Institutions? Most often, the scientist is forced to purchase Expensive & / or huge Liquid Scintillation counters, which may or may not be required for his particular application. In majority of the instances, the scientist or the end user is usually unaware of the products or choice of LSC that are available & / or is being made to believe / accept the LSC by the so called technical talk on Figure of Merit, Background, Sample Capacity, Brand Name etc. and by the References of usage of multi vial readers in publications or technical literature. 2) Are Multi Vial LSCs economical for Research Institutions? No, Multi Vial LSC are extremely huge, occupy huge lab space and are very expensive. Even the cost of maintenance is high. Triathler MLT, on the other hand has the capability of counting both Beta & Gama emitters and is the LSC of choice of Research Institutions. For example, in a developing country like India, where the resources are meagre, a single vial LSC will meet all the requirements of Life Science Applications. This would reduce the cost of purchase of LSC by more than 60% and an additional saving of money in the maintenance of the equipment.

2 3) What are the main applications of LSC in estimation of Radioactivity? Let us have a cursory look at the need for quantification of Radio nuclides. Radioactive labelled probes are mainly used in the following applications: APPPPLLIICCATTIION Life Sciences Drug Discovery Chemistry Geology Nuclear Industry DESSCCRIIPPTTIION Molecular Biology application like Insitu Hybridization, DNA Finger Printing, Mol Diagnostics etc.. or Biochemical applications like analysis of Enzymes & its pathways etc.. Mainly for the Pharmaceutical Industry & CRO s for Screening of NCE s & ADME applications For Physio- Chemical Investigation of Chemical Reactions etc Hydrological pathways For estimation of Tritium, Sr, U, Am, Pu and other Alpha & Beta emitters 4) What are the main radio nuclides that are used in Research? Soft Betas, ( Exception is P32 ) are most often employed for Life Sciences Applications. Some of the most commonly used Radioisotopes are: ISOTOPE 32P 33P 3H 14C 35S FEATURES Has a E Max of 1.72 MeV and is the most energetic beta emitter that is used in Life Sciences applications. 32P is mainly used for Nucleotide analysis. Conventionally 32P is estimated using a vial Reader. A fraction of the gel is dissolved using Sodium Hypochlorite solution and suitable Cocktail is added for quantification of 32P that is incorporated in the nucleotides. The same procedure can be carried out in a 96 well Isoplate using a LSC Plate Reader. Has a E Max of 249 KeV and is mainly used for Nucleotide Analysis Drying the sample increases the sensitivity of detection. Has a E Max of 156 KeV and is a popular beta emitter that is used in life Sciences. 35S : Has a E Max of 167 KeV and is mainly used for Protein & Peptide related assays. 125 Iodine Primarily a Weak Gama Emitter with E max 35.5 KeV. The Compton electrons that are emitted are detected using Cocktails in LSC mode. 125 Iodine is mainly used in RIA for estimation of Thyroid Hormones etc.. 5) What type of Liquid Scintillation counters (Vial or Plate Type ) are more suited for counting radioactive samples for Life Sciences applications?

3 For most of the biological assays like Proliferation assays or Incorporation assays, 1 uci (2,220,000 DPM) of activity (Labelled Radionuclide) is added to the culture / assay. The specific activity & / or amount is determined or optimized so that after harvesting a minimum of 10,000 counts are observed. This explains the reason for Back Ground or Figure of Merit being not applicable to biological applications. LSC Plate Readers provide good counting efficiency & performance while counting Filter plates or using Isoplates and the results correlate with the values of a vial reader. 6) What are the methods that are used for Separating Bound vs Unbound Radio-ligands? Separation of bound (incorporated) and unbound (unincorporated) radio-ligand is often the most critical step in receptor binding assays or Cell proliferation assays. Separation can be affected either by: Filtration Centrifugation Chromatography Electrophoresis etc. 7) What are the advantages of using Filtration for separation? Filtration using filtration manifold / Harvester is the most widely used technique and some of the advantages are: Reduction in number of Liquid Handling steps (Less exposure to radioactivity) Substantial reduction in separation time Ease of sample preparation & counting Filter plate or mats can be directly counted in a plate reader there by eliminating the need for punch & deposit in a vial for counting in a vial reader. Automation compatible for high throughput screening For receptor binding assays, kd is inversely proportional to separation time and filtration is the Only preferred method of separation. 8) What are the other advantages of Chameleon LSC Plate Reader? Chameleon LSC Plate reader can also further help you in reduction of: Sample Volume Requirement Typically, 20 uls of sample uls of cocktail totaling 200 uls is used while counting Liquid samples using Isoplate. For Filter bottom plate, only 20 uls of Cocktail is used for counting. This is in comparison with 7-10 ml of sample / cocktail volume for a vial reader. Reagent volume (more assays can be done with the same kit there by reducing the cost per sample) Reduction in waste generation & disposal. 9) Can Gama emitters be quantified in a Liquid Scintillation counters? Only weak Gama emitters like 125 Iodine ( 51 Cr ) which also emit Augur electrons can be quantified in a LSC. Note that Gama radiation is not detected but the Augur electrons are detected and quantified. Other conventional Gama emitters like 60 Co, 129 I, etc.. cannot be quantified in a LSC but require Gama counter with impregnated NaI detector for detection & quantification.

4 10) What is Quench? How does it affect Liquid Scintillation counting? Quench is defined as Reduction in Counting efficiency. Chemical Quench: Reduction in the transfer of energy from Beta particle to Scintillant, thereby reducing the no. of Scintillations produced. This results in shift of energy to Lower Channels & Reduction in the number of counts (Counting efficiency).chemical Quench is not affected by volume. Color Quench: Absorption of the emitted Scintillations by the colored components, thereby reducing the no. of counts detected. Color quench varies with sample volume. 11) What are the effects of Quench in counting samples in LSC? Shift of energy spectrum to lower channels Reduction in Counting efficiency 12) How is Quench estimated using Triathler LSC? Quench Parameter (QP) indicates the relative light production and is the centre of gravity of the energy spectrum. QP & Counting efficiency rarely have linear relation. Triathler & Chameleon use either Transformed Parabolic Regression or Segmented Linear Interpolation to form Quench Curve. Based on the Qp obtained from the sample, counting efficiency is extrapolated from the Quench Curve. DPM or corrected CPM is derived by dividing the CPM with counting efficiency. 13) What are the methods for estimation / estimation of variable quench? The different methods for Variable Quench Correction: Using Internal Standards Using Quench Curve obtained using Quench Standards ( Internal ) Using External Gama Source for Obtaining Quench Curve & QpE Instant DPM Method. 14) What is Triple to Double Coincidence Counting Ratio? TDCR, Triple to double coincidence ratio method is an absolute activity method specially developed for counting pure beta and pure EC - emitter s activity determination, in which the detection efficiency is calculated from a physical and statistical model of the photon distribution emitted by the scintillation source

5 15) How is Quench Correction carried out in 300 SL TDCR Liquid Scintillation counter? In addition to the above mentioned method, 300 SL employs Triple to Double Coincidence Counting Ratio to estimate Quench in sample. The Free Parameter which is a measure of Quench, is proportional to TDCR. TDCR value is arrived at, by the 300SL. CPM divided by TDCR gives Corrected DPM values within +/- 15% accuracy. 16) What are the available Liquid Scintillation counters which can be used for Alpha beta separation? Tricarb Series 2900 TR, 3100 TR, 3170 TR / SL, Quantulus 1220, Triathler LSC, 300 SL TDCR LSC are the available LSC s with optional Alpha beta discrimination. Note: Tricarb & Quantulus are the registered trademarks of Perkin Elmer USA while Triathler & 300 SL are the registered trademarks of Hidex Oy Finland. 17) Is Pulse Decay Discrimination the only method for Alpha beta separation? Tricarb Series of equipment use Time Resolved Liquid Scintillation Counting for detection of radio isotopes and use Pulse Decay Discrimination for Alpha beta separation. While 300 SL TDCR LSC & Triathler LSC (Quantulus 1220??) use Pulse Length Index for Alpha beta separation. Pulse Length Index for Alpha Beta separation: Alpha & beta counts can be differentiated in 300 SL TDCR LSC & Triathler LSC by using Pulse Length Index and separation efficiency is nearly 100%, depending on the type of sample & counting conditions. 18) What is Pulse Length Index principle that is used in Alpha beta separation? The principle of PLI using in Alpha beta separation, in brief, is as follows: Alpha particles produce much denser ionization than betas, and correspondingly much higher ionization quench. Therefore, light output per energy from alpha scintillations is actually near 10 times lower than from beta scintillations. In other words, pulse from 6 MeV alpha comes near the same MCA channel as pulse from 600 kev beta. Alpha pulses are longer than the beta Intensity / counts 2D Spectrum of Uranium in TBP extract Energy Pulse Height pulses. This principle is used for differentiating Alpha pulses from Beta pulses. For example a Alpha Pulse of 6 MeV, which appears appr at 600 kev, is differentiated by 600 kev beta pulse depending on the length of the pulse. In alpha beta separation, length of the pulse (alphas > beta) is used to differentiate between Alpha & Beta Pulses In the 2D Spectrum, X Axis represents Energy; Y axis represents the amplitude & Z axis intensity.2d spectrum gives a graphical / pictorial representation of alpha beta separation and by viewing the 2D spectrum it is very easy to optimize the PLI for better separation.