Frontispiece Top: An autoradiogram showing the deposition of iodine-131 in the kidney of a rat. Iodine-labeled pro­ teins were injected intravenously into a rat which was sacrificed a few hours later. The blackened areas show the deposition of the iodine, and presumably the pro­ teins, in the glomeruli. The wet-mounting technique with an NTB plate was used with metanil yellow stain­ ing after photographic processing. Courtesy P. J. Fitzgerald (unpublished), Sloan-Ket- tering Institute. Bottom : An autoradiogram showing the deposition of sulfur-35 in the skin of a newborn mouse. One mc of inorganic sulfur-35 was injected intraperitoneally into the mother 24 hours before birth. The autoradio- graphie evidence is the random distribution of grains over three cell nuclei. These are probably mast cells. The developing hair follicles show no uptake. The permeable-base stripping film technique was used with haematoxylin and celestin blue staining after photo­ graphic processing. Courtesy A. Glücksmann, Alma Howard, and S. R. Pelc, Radiotherapeutic Research Unit, Hammersmith- Hospital, London. AUTORADIOGRAPHY IN BIOLOGY AND MEDICINE GEORGE A. BOYD Director, Arizona Research Laboratories, Phoenix, Ari­ zona. Formerly, Professor of Biophysics, University of Tennessee, and Senior Scientist, Oak Ridge Institute of Nuclear Studies ACADEMIC PRESS INC., PUBLISHERS NEW YORK, 1955 COPYRIGHT 1955, by ACADEMIC PRESS INC. 125 EAST 23RD STREET NEW YORK 10, N. Y. NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS. Library of Congress Catalog Card Numker: 54-11051 PRINTED IN THE UNITED STATES OF AMERICA DEDICATED TO DR. W. W. STEFFEY Preface Autoradiography in biology and medicine is a marriage of the arts of histology and photography. As in the fine arts, there are as many ways of doing autoradiography as there are doers. There are, however, scien­ tific pathways to follow in learning the art. This book is divided into three sections. In the first section is given much of the background for the scientific approach to the art given in the second section. The many techniques given in the second section are in "cook-book" style and can be followed explicitly by the laboratory technician. There is no guarantee that a technique will work in every laboratory, in every part of the country, or for all materials to be used. It is almost impossible for the originator of a technique to recognize some essential steps and transfer them to me by conversation and I to the reader through the printed page. I have attempted to elicit, from the authors, all the little details which we so often unconsciously perform in the laboratory; those details which we sometimes have sound, but intuitive, reasons for doing yet forget to include in a published paper. Sometimes these unconscious acts are mere habit. They may make for success of a technique. This is the personal art : the reason some are successful where others are not. I have written this book for the researcher who has never done auto­ radiography. The technique to use will be indicated by the nature of his investigation. A study of the first section will help him decide on the materials to be used in the experiment to obtain meaningful information. It will be obvious that many of the steps of a technique can be varied. None of them are sacred. Rather, they are diving boards from which you can take a plunge into the pool of variables and come up with your own pet combination—a new technique. The quantitative aspects of autoradiography have not been included. The book was growing to unmanageable proportions. Also, much good vii viii PREFACE work in this field is in process and has not been published. A section on quantitative autoradiography at this time would be premature. The third section is an extensive bibliography handled in, I believe, a novel manner. This is discussed in detail in the Introduction to the Bibliography. I owe my interest in autoradiography to Professor William Bale of the University of Rochester who suggested I investigate its potentialities in the study of polonium distribution in tissue sections. I am grateful to him, even though the study of the technique temporarily diverted me from my earlier interest of radiocolloids in tissues. The decision to write a book came through a suggestion of Dr. Kurt Salomon, also of the University of Rochester. I thank him for this, and am sure I shall feel more kindly toward him after corrections are made and the presses start. GEORGE A. BOYD Acknowledgments I wish especially to thank Dr. Harry Blair and Dr. J. Newell Stannard of the University of Rochester for their interest in the work. It is a pleasure to acknowledge the interest shown in the writing by Dr. C. L. Cornar, Dr. R. T. Overman, and Dr. M. Brucer. I am especially grateful to Dr. Overman for his encouragement and kindly criticism during the preparation of the manuscript. I owe a special debt of gratitude to Dr. D. R. Smith who helped me set up a punch card system, without which I could not have written the book. Assistance in searching was generously given by Mr. R. A. Schlueter, Chief Librarian of ORINS. The Misses Marion Garber and Louise Markel and my secretary, Mrs. Richard Broad, did the painstaking job of checking the literature references. Over a period of several years, various members of the Eastman Kodak Company have been most generous with their time in discussing several phases of this book, reading some of the chapters, and advising on the presentation of material. It should not be inferred that they neces­ sarily put their stamp of approval on my statements. Any errors of photo­ graphic theory and facts are mine in spite of their excellent teaching. For making this a better book, I want to thank Drs. Cyril Staud, John Spence, Julian Webb, Herman E. Seeman, T. H. James, and B. H. Carroll, all of the Eastman Kodak Research Laboratories, Mr. William Swann and Mr. R. Tobey of the Sales Department, and Mr. William Cornwell, Editor of Medical Radiography and Photography. I am indebted to Mr. John Barione of Saint Norbert College who read almost the entire manuscript. He looked at it through the eyes of an histologist, and much improved the presentation. The publishers have been most considerate and helpful. And last I wish to thank the one in the background who furnished the inspiration and the understanding of my moods of elation when the writing went well and of depression when it went sour, who deflected the romping and yelling children when I wished to write, and who was my chief critic : Carolyn, my wife. îx CHAPTER I Introduction 1. HISTORICAL On Monday, January 20, 1896, the French Academy of Sciences met in Paris as usual for the reading of papers and the discussion of the latest scientific events. Henri Becquerel attended as was his custom and as had been the custom of his father and grandfather. All three were physicists and had held the research post of Professor of Physics at the Museum of Natural History. Henri also held the teaching post at the Ecole Polytechnique. His father, Edmond, and Henri had studied fluorescence. With this background Henri attended the meeting where he was to hear exciting news. Henri Poincaré showed the X-ray pictures sent by Rontgen from Vienna and discussed the paper which Rontgen had sent to press December 29, 1895. Poincaré suggested that the X-rays might be a portion of the fluorescence radiation which Rontgen had seen on the wall of his glass X-ray tube. This set Becquerel thinking. The next day he started his investiga­ tions of several fluorescing materials but most carefully of a flat crystal of potassium uranyl sulfate which he had made. He wrapped a photo­ graphic plate in black paper, placed a crystal on top, and exposed the combination to sunlight. On developing the plate after several hours exposure, he was pleased to see a faint outline of the crystal—the first autoradiogram, although unrecognized. Becquerel thought the X-ray­ like radiation was associated with the fluorescence. He continued his experiments and showed that the blackening was always associated with, and only with, salts or solutions containing uranium, even though fluorescence was not always present. In the same year he showed that pure uranium metal produced the blackening. Nevertheless, he con- 3 4 THEORY tinued to think that fluorescence, even though not always seen, was in some way connected with the phenomenon. Thus, through serendipity, the first radioactivity autoradiogram was made but not recognized. Becquerel's inability to give up the idea of fluorescence prevented him from suspecting an entirely new phenom­ enon. Although he discovered the first raw facts of radioactivity, he failed to understand and interpret them. It remained for Marie Curie, in the Spring of 1898, to establish by ionization measurements the rela­ tion of radiation quantity with the quantity of the uranium element. Thus, she was the first to recognize radioactivity.1 It seems apropos to digress for one paragraph to consider the nature of discovery, since autoradiography can easily produce raw facts which, in themselves, do not constitute discovery. I shall let Claude Bernard comment :2 "We usually give the name of discovery to recognition of a new fact : but I think that the idea connected with the dis­ covered fact is what really constitutes the discovery. Facts are neither great nor small in themselves. A great discovery is a fact whose appearance in science gives rise to ideas shedding a bright light which dispels many obscurities and shows us new paths. There are other facts which, although new, teach us but little; they are therefore small discoveries. Finally, there are new facts which, athough well observed, mean nothing to anyone; they remain, for the time being, detached and sterile in science; they are what we may call crude or raw facts." Autoradiography was first intentionally used for locating radio­ activity in minerals (12). The first published ( 1904 ) biological appli­ cation was that of E. S. London of the Imperial Institute of Experi- 11 am indebted to Professor Alfred Römer of St. Lawrence University, Canton, New York, for permission to see the manuscript of Chapter 2 of his forthcoming book, "The Development of Atomic Physics," from which I have drawn heavily for the facts of the first four paragraphs. For a more detailed and excellently documented account of Becquerel's investigation, see Professor Romer's book. 2J. M. D. Olmsted and E. H. Olmsted, Claude Bernard and the Experi­ mental Method in Medicine, p. 6. Henry Schuman, New York (1952).