AN ABSTRACT OF THE THESIS OF David Lee Willis for the Doctor of Philosophy in General Science (Name) (Degree) (Major) Date thesis is presented May 10, 1963 Title RADIOTRACER METHODOLOGY IN BIOLOGICAL SCIENCE Abstract approved (Major professor) The use of radioactive isotopes as tracers in biological sys- tems has become widespread since the close of World War II. Proper use of radiotracers requires a fundamental understanding of the physi- cal nature of radioactivity, the characteristics of ionizing radiation, and the various methods available for measuring radioactivity. More importantly, the investigator employing radioisotopic tracers must be familiar with the methodology involved in design of radio - tracer experiments, the preparation of radioactive samples for assay, and the problems inherent in analyzing data from radiotracer experi- ments. The purpose in the preparation of this thesis was to present a summary of the essential concepts and information needed by the biologist who desires to make use of radiotracer methods in his in- vestigations. The thesis is set forth in the form of an introductory text, suitable either for class or individual use. The presentation is divided into three major sections: (I) the text proper, covering the principles of radiotracer rnethodology, (?) a set of basic labora- tory exercises, intended to farniliarize the user with procedures in detecting and characterizing radioactivity, and (3) a selection of typical radiotracer experiments illustrating applications in varior.zs fields of biological science. This latter secti.on is thought to be parti- cularly valuable in that it furnishes step-by-step examples of design and execu.tion of typical radiotracer experirnents " In view of the fact that liquid sci.ntillation counting has recently come into widespread favor arnong biologists using tritium and carbon-14 labeled tracer compounds and yet no comprehensive moilo- graph is available on the subject, particular attention has been de- voted to this assay rnethod. A rnost extensive bibliography covering both the preparation of sarnples for liquid scintillation counting and the operating characteristics of the counter assembly is included" In addition, one of the laboratory exercises deals with the practical operation of a liquid scintillation counter" Other aspects of radiotracer methodology that are treated are the safe handling of radioisotopes, the proper design of radiotracer laboratory facilities, and the statistical analysis of counting data. since the biologist commonly secures his radiotracer compounds from commercial radiochemical suppliers, a chapter on the methods of primary radioisotope production and the preparation of labeled compounds has been included as background information. The re- cently popular technique of tritium labeling by gas- exposure (the Wilzbach method) has also been discussed in this connection. Although the emphasis in this presentation has been restricted primarily to the application of radiotracers to research in the biologi- cal sciences, the coverage is broad enough to be of value to investiga- tors in other fields. Copyright by DAVID LEE WILLIS 1963 RADIOTRACER METHODOLOGY IN BIOLOGICAL SCIENCE by DAVID LEE WILLIS A THESIS submitted to OREGON STATE UNIVERSITY in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY June 1963 APPROVED: Professor of Chemistry In Charge of Major rman of Departmerìt of Gene Scien Dean of G aduate School Date thesis is presented MAY \O, 19C.3 Typed by Jolene Wuest ACKNOWLEDGMENTS I would like to express my sincerest appreciation to my major professor, Dr. Chih H. Wang, without whose encouragement and ad- vice this work would never have been attempted, nor completed. Other members of the Radiotracer Laboratory in the Science Research Institute at Oregon State University, both graduate students and staff, deserve my thanks for willingly sharing their time and ex- perience. Finally I want to express my gratitude to my wife, Earline, for untold hours spent in typing, proofreading and generally assisting in the completion of this volume. TABLE OF CONTENTS Page INTRODUCTION 1 PART ONE -- PRINCIPLES OF RADIOTRACER METHODOLOGY CHAPTER 1 ATOMS AND NUCLIDES 10 A. General Structure of the Atom 10 B. The Nucleus 13 C. Nuclides and Isotopes 15 D. Stable Nuclides 17 E. Unstable Nuclides (Radioisotopes) 20 1. Naturally occuring radioisotopes 21 2. Artificially produced radioisotopes 24 a. By particle accelerators 24 b. By nuclear reactors 26 BIBLIOGRAPHY 30 2 THE NATURE OF RADIOACTIVE DECAY 32 A. Radionuclides and Nuclear Stability 32 B. Types of Radioactive Decay 34 1. Decay by negatron emission 36 2. Decay by positron emission 37 3. Decay by electron capture 39 4. Internal conversion 41 5. Isomeric transition 41 6. Decay by alpha particle emission 41 C. Rate of Radioactive Decay 42 1. The decay constant 43 2. Half -life 48 3. Practical decay considerations 53 4. Composite decay 55 5. Average life 58 D. The Standard Unit of Radioactivity -the Curie 59 E. Specific Activity 62 BIBLIOGRAPHY 64 CHAPTER Page 3 CHARACTERISTICS OF IONIZING RADIATION 65 A. Alpha Particles 66 1. Energy 66 2. Half -life and energy 67 3. Interaction with matter 70 a. Excitation 70 b. Ionization 70 c. Specific ionization 71 4. Range 73 a. Determination 73 b. Range- energy relations 73 c. In other materials 74 5. Practical considerations 76 B. Beta Particles 77 1. General nature 77 a. Negatrons and positrons 77 b. Conversion electrons 78 2. Energy of beta decay 79 a. Spectral distribution of energy 79 b. The neutrino and beta decay 82 c. The Fermi theory of beta decay 84 3. Interaction with matter 85 a. Modes of interaction 85 b. Specific ionization 85 4. Range 87 5. Practical considerations 89 a. In detection 89 b. Biological hazards 91 G. Gamma Rays 92 1. Nature and source 92 a. Electromagnetic nature 92 b. Source of gamma emission 93 c. X -rays 95 2. Mechanisms of interaction with matter 95 a. Nuclear transformation 97 b. Mössbauer effect 97 c. Bragg scattering (diffraction) 97 d. Photoelectric effect 98 e. Compton effect 98 f. Pair production 99 3. Absorption relations 101 a. Linear absorption 101 b. Mass absorption 104 Page c. Half thickness 106 d. Dependence on gamma energy and absorber density 109 4. Practical considerations 114 D. Summary 115 BIBLIOGRAPHY 116 CHAPTER 4 MEASUREMENT OF RADIOACTIVITY: GENERAL CONSIDERATIONS AND THE METHOD BASED ON GAS IONIZATION 117 A. Absolute Counting vs. Relative Counting 117 1. Types of radioactivity measurements 117 2. Considerations in relative counting 118 B. Basic Mechanisms of Radiation Detection 120 1. Gas ionization 120 2. Scintillation 120 a. In a solid fluor 120 b. In a liquid fluor 121 3. Autoradiography 122 C. Gas Ionization 122 1. Without gas amplification 124 a. Ionization chambers in general 124 b. Lauritsen electroscopes 130 c. Vibrating -reed electrometers 132 2. With gas amplification 135 a. The nature of gas amplification 135 b. The proportional region 138 c. The limited proportional region 139 d. The Geiger -Mueller region 140 (1) Dead Time 141 (2) Quenching 142 3. Proportional detectors 144 a. Construction 144 b. Operating characteristics 147 4. Geiger -Mueller detectors 149 a. Construction 149 b. Operating characteristics 154 5. Summary of gas ionization detectors 156 BIBLIOGRAPHY 158 CHAPTER Page 5 MEASUREMENT OF RADIOACTIVITY BY THE EXTERNAL - SAMPLE (SOLID)SCINTILLATION METHOD 1 60 A. Basic Facets of the Scintillation Phenomenon 160 B. External - sample (Solid) Scintillation Detectors 162 1. Mechanism of external - sample scintillation detection 162 a. Energy conversion in the fluor crystal 164 b. Energy conversion in the Photomulti- plier 165 c. Proportionality of energy conversion 166 d. Other advantages 168 e. Photomultiplier thermal noise 168 f. Gamma ray spectrometry 169 2. Components of external sample scintillation detectors 170 a. Detector housing 170 b. Fluor crystals 172 c. Photocoupling 174 d. Photomultiplier tubes 175 3. Operating characteristics of external - sample scintillation detectors 178 a. Effect of photomultiplier potential 179 b. Effect of amplifier gain 181 c. Gamma energy dependence of detection efficiency 181 BIBLIOGRAPHY 183 6 MEASUREMENT OF RADIOACTIVITY BY THE INTERNAL - SAMPLE (LIQUID) SCINTILLATION METHOD 1 85 A. Mechanism of Internal - Sample Scintillation Detection 1 87 1. Energy conversion steps in the fluor solution 1 87 2. Comparative examples of energy transfer efficiency for C14 and H3 beta particles 1 89 B. Evaluation of the Internal - Sample Scintillation Method 1 93 1. Advantages of liquid scintillation counting 193 Page 2. Problems inherent in liquid scintillation counting 1 95 a. Photomultiplier thermal noise 195 b. Counting sample preparation 197 c. Quenching 198 C. Components of an Internal- Sample Scintilla- tion Counter 201 1. The operating mechanism 201 2. The detector assembly 202 a. Optical components 202 b. Fluor solution components 203 (1) Primary solvents 204 (2) Secondary solvents 205 (3) Primary solutes 207 (4) Secondary solutes 209 D. Special Types of Liquid Scintillation Detectors 210 1. Large volume external - sample detectors 210 2. Continuous flow scintillation detectors 211 E. Operating Characteristics of Internal - Sample Scintillation Counters 213 1. Selection of optimal counter settings 214 a. Effect of photomultiplier gain and "window" settings 214 b. Balance point operation 215 c. Flat spectrum operation 217 2. Determination of counting efficiency 220 a. Internal standardization method 221 b. Dilution method