RESULTS YOU CAN COUNT ON

Transcription

1 RESULTS YOU CAN COUNT ON Liquid Scintillation Counting Principle and the Application in Biological Research PerkinElmer

2 liquid scintillation counting β particle Tri-Carb family of liquid scintillation analyzers TopCount NXT plate counters 3 H 14 C 32 P MicroBeta 2 plate counters Quantulus Liquid Scintillation Spectrometer 35 S 2

3 Isotopic Properties Isotope Half-Life Maximum Theor SA Energy Emitted 32 P 14.3 Days 9120 Ci/mmol B-1.71MeV 33 P 25.4 Days 5000 Ci/mmol B-0.25MeV 35 S 87.4 Days 1488 Ci/mmol B-0.167MeV 3 H Years 29 Ci/mmol B-0.019MeV 14 C 5,730 Years 62 mci/mmol B-0.156MeV 3

4 Radioactivity: Simulated Decay of 32 P Phosphorous 32 Sulfur 32 Sulfur 32 Sulfur 32 Phosphorous 32 Phosphorous 32 Phosphorous 32 0 days ~14 days ~28 days ~42 days 4

5 Radioactivity Units: Measured through Decay Becquerel (Bq) IUPAC 1 Becquerel is defined as 1 disintegration per sec (dps) Curie (Ci) most common 1 curie is defined as 3.7 X disintegration per sec (dps) To convert from Bq to Ci and vice versa: 1Bq = 2.7X10-11 Ci= 1 dps = 60 dpm 1Ci = 3.7X10 10 Bq= 3.7X10 10 dps = 2.22X10 12 dpm dpmis the abbreviation for disintegration per min. This unit is directly related to the counts that customers measure by their counter. 5

6 Concentration Concentration refers to the amount of material (solute) dissolved in a given volume of liquid (solvent) There are two ways of expressing concentration when dealing with radioactivity: 1. Radiochemical concentration (mci/ml or µci/ml) = total amount of radioactivity per a certain volume 2. Molar concentration (µm) = actual concentration of the labeled product (example, amount of labeled nucleotide) 6

7 Radiochemical vs. Molar Concentration Radiochemical and molar concentration can dictate what a customer purchases The radiochemical concentration and the molar concentration have a direct linear relationship The SA are the same in the product below, but the radiochemical and molar concentrations are different UTP ( 32 P) SA Ci/mmol CONC mci/ml Molar Conc. BLU507X 800Ci/mmol 10 mci/ml 12.5µM BLU507T 800Ci/mmol 20 mci/ml 25µM BLU507C* 800Ci/mmol 40 mci/ml 50µM * BLU507C is no longer available 7

8 Specific Activity Definition: The amount of radiolabeled mass in a sample Can be abbreviated as SA Each isotope has a maximum theoretical specific activity Measured in Ci/mmol All have the same total mass of dctp. Only the amount of labeled 32 P (SA) differs. 8

9 Why choose a certain Specific Activity Value? One of the key factors customers use to determine which product they will buy 32 P alpha dctp Specific Activity Concentration BLU613A 800Ci/mmol 10 mci/ml BLU613H 3000Ci/mmol 10 mci/ml BLU613Z 6000Ci/mmol 20 mci/ml Assay Efficiency and Detection Method Criteria for choosing which SA they want Some deleted products (low sellers) can be substituted by a different specific activity product 9

10 PerkinElmer Radiochemicals Toolbox 10

11 Application Overview of PerkinElmer RAD business Isotope Application 3 H Cell proliferation 51 Cr Cell cytotoxicity (Chromium release) 3 H, 32 P Reverse transcriptase 125 I FlashPlates (RIA, binding, etc.) 125 I, 3 H SPA (Scintillation proximity assay) 32 P Labeled Probes 3 H Receptor Binding Various Lab Surveys 125 I (Radioimmunoassay) 125 I Binding assays 51 Cr Chromium release assay Various High energy gamma emitters Various Multiple isotope analysis (3 or more) Various Microspheres 111 In-, 131 I, etc. Radiotherapeutics C-11, F-18, O-15, Sc-46, etc. PET 11

12 Why Liquid Scintillation Counting? Utility - Many Nobel laureates in Biology for the past 50 years used radiotracers and LSC Versatility - applicable to all forms of nuclear decay emissions: a, -B, e-, g Sensitivity Specificity 12

13 Liquid Scintillation Counter 13

14 The principle of liquid scintillation counting-step1 14

15 The principle of liquid scintillation counting-step2 15

16 The principle of liquid scintillation counting-step3 16

17 Liquid Scintillation Counting Theory β PMT Key! This process is a linear relationship, therefore # photons is directly proportional to β- energy generated from the decay event 17

18 Quench beta decay solvent fluor light physical chemical colour DPM = actual number of nuclear decay events (goal) CPM = number of decay events detected by the liquid scintillation counter 18

19 Equation for Calculating DPM Net CPM DPM = Counting Efficiency Unknown Variable Accurate results depend on good sample preparation first, quality instrumentation second. Model A307 Oxidizer 19

20 Correction methods: tsie tsie= transformed Spectral Index of Sample The unknown sample is first measured without external standard. Spectral information is stored Energy spectrum measured in the presence of external standard The average pulse height in the Compton-spectrum from the external standard determined by the instrument tsie Value of tsie is a function of: Type and degree of quenching 20

21 tsie: Example of standard curves for 3 H and 14 C 21

22 Quality control of measurements Reproducibility / precision Statistical analysis Accuracy / absence of systematic sources of error Knowledge of instrument design, function, and correct usage Knowledge of physical processes in instrument 22

23 Optimizing Counting Consider quench and other counting interferences Homogeneous Sample Clear, not milky No phase separation 4π counting geometry Remember: the detector is in the vial Choose the appropriate cocktail and vial ŁGOAL: determine actual amount of isotope in the sample 23

24 Chemical quenching and color quenching Both phenomena lead to a reduction of pulse height spectra for both sample and external standard are displaced towards the left The mechanism for the two types of quenching is different different standard curves Precautions: Avoid color quenching as far as possible (bleaching of samples etc.) Use same standard curve for all chemical quenchers Different standard curves for classical and safe cocktails CHCl 3 is often used as standard chemical quencher 24

25 Practical activity determination in LSC Measurement of a series of samples, incl. background and standard samples Background samples Standard samples instrument -standards (calibration, test of reproducibility) Experiment standards Unknown samples Grouping according to nature of experiment or assay 14 C standard 3 H standard SNC Background standard 25

26 Instrumental determination of instrument performance Establish performance criteria to set baselines IPA screens from a Tri-Carb with QuantaSmart software 26

27 Collecting the Sample Wipe test Take samples using a S motion while rotating the cotton swab. Also shown, collecting a sample using a chem wipe. 27 From The University of Texas

28 Preparing the Samples Wipe test Take cut cotton swab and place in tube Fill tube with approximately 1mL of cocktail Close tube 28 From The University of Texas

29 Loading the Samples Wipe test Place closed tube into vial holder Tab position indicates vials to be read. Tab position indicates vials will not be read Load rack into LSC to run wipe test 29 From The University of Texas

30 Wipe test 30

31 31 Questions & Feedbacks