X-Ray Microanalysis in Biology PDF
Preview X-Ray Microanalysis in Biology
X-RAY MICROANALYSIS IN BIOLOGY X-RAY MICROANALYSIS IN BIOLOGY edited by M. A. Hayat, Ph.D. Professor of Biology Kean College of New Jersey Union, New Jersey M © University Park Press 1980 Softcover reprint of the hardcover 1st edition 1980 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. First published in the USA 1980 by University Park Press, Baltimore. Published in the UK 1981 by Scientific and Medical Division MACMILLAN PUBLISHERS LTD London and Basingstoke Companies and representatives throughout the world. ISBN 978-1-349-06176-1 ISBN 978-1-349-06174-7 (eBook) DOI 10.1007/978-1-349-06174-7 Contents Contributors ......................................... vi Preface ............................................. vii 1. Principles and Instrumentation.. . . . . . . . . . . . . . . . . . . . . . . 1 Alan T. Marshall 2. Preparation of Specimens: Changes in Chemical Integrity ...................... 65 A. John Morgan 3. Frozen-Hydrated Bulk Specimens ...................... 167 Alan T. Marshall 4. Frozen-Hydrated Sections ............................ 197 Alan T. Marshall 5. Sections of Freeze-Substituted Specimens .............. 207 Alan T. Marshall 6. Influence of Specimen Topography on Microanalysis .................................... 241 F. D. Hess 7. The Skeletal Muscle ................................. 263 Michael Sjostrom 8. Liquid Droplets and Isolated Cells ..................... 307 Joseph V. Bonventre, Kristina Blouch, and Claude Lechene 9. Quantitative X-Ray Microanalysis of Bulk Specimens ....................................... 367 F. Duane Ingram and Mary Jo Ingram 10. Quantitative X-Ray Microanalysis of Thin Sections ......................................... 401 Godfried M. Roomans Author Index ....................................... 455 Subject Index ....................................... 467 Contributors Kristina Blouch, Biotechnology Resource in Electron Probe Microanalysis, 45 Shattuck Street, Harvard Medical School, Boston, Massachusetts 02115 Joseph V. Bonventre, Biotechnology Resource in Electron Probe Micro analysis, 45 Shattuck Street, Harvard Medical School, Boston, Massa chusetts 02115 F. D. Hess, Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 F. Duane Ingram, Department of Physiology and Biophysics, The Univer sity of Iowa, Iowa City, Iowa 52242 Mary Jo Ingram, Department of Physiology and Biophysics, The Univer sity of Iowa, Iowa City, Iowa 52242 Claude Lechene, Biotechnology Resource in Electron Probe Microanalysis, 45 Shattuck Street, Harvard Medical School, Boston, Massachusetts 02115 A. T. Marshall, Department of Zoology, LaTrobe University, Bundoora, Victoria, Australia 3083 A. J. Morgan, Department of Zoology, University College, P. 0. Box 78, Cathays Park, Cardiff, Wales, U.K. Godfried M. Roomans, The Wenner-Gren Institute, University of Stock holm, S-113 45 Stockholm, Sweden Michael Sjostrom, Department of Anatomy, University of Umea, S-901 87 Umea, Sweden vi Preface X-ray microanalysis (electron-probe x-ray analysis, electron probe microanalysis, or analytical electron microscopy) is a relatively new method of elemental analysis at the ultrastructural level. The major contribution of x-ray microanalysis to cell biology is its ability to correlate morphological appearance with chemical composition. Such in formation is important in understanding the mechanisms that control various cellular processes. Ionic regulation of cellular processes such as muscular contraction, proto plasmic movement, hormone secretion, chromatic condensation, and mitosis is well known. No other method is presently available that can provide an immediate correla tion between the structure of a submicroscopic cellular component and its biochem istry. Rapid progress in the application of x-ray microanalysis to the biomedical area has been made during the past few years. This has been possible partly because x-ray microanalysis has been coupled with scanning, transmission, and scanning transmis sion electron microscopy. Ion distribution in normal as well as in pathological cell sys tems of both plant and animal specimens can be studied routinely by using these in struments. Major improvements have been made in many aspects of methodology, includ ing the size of the specimen, the sensitivity, and the range of detectable substances. In fact, the range of detectable substances is almost limitless, and even elements of low atomic number can be studied by using secondary ion emission analysis, laser, or Auger electrons. In addition, stable or radioactive isotopes can be separated, and cathodoluminescence allows the study of certain types of molecules. It is now possible to achieve an analytical spatial resolution of 20-30 nm. Since the spatial resolution and sensitivity of x-ray microanalysis are sufficient to. permit measurement of element concentration in a single cell as well as in an organelle, this method has become an important complement to conventional scanning and trans mission electron microscopy. Identification and localization of main elements of the Periodic Table in the specimen are accomplished with a specificity and sensitivity never attained in the past. The limiting factor in x-ray microanalysis of biological specimens is the difficulty in preparing the specimen rather than in the performance of the instrument. Another limiting factor is specimen damage caused by the electron beam. Ideally, the amount and location of elements in the prepared specimen should be the same as in the living specimen. However, this feat is difficult, if not impossible, to accomplish. In any in terpretation of the data obtained by using x-ray microanalysis, specimen preparation conditions must be taken into account. For example, specimens differ in their elemen tal composition when prepared by air drying as compared with various freezing tech niques. The best approach to achieve a valid x-ray microanalysis of naturally occur ring elements in biological specimens seems to be the use of hydrated, unfixed, ultra- vii viii Preface thin frozen sections, provided the extent of ion diffusion is understood and possibly reduced, and the preservation of structural details is improved. Cryopreparation is neeessary for determining the storage or binding sites of physiologically active ele ments. In the sections of quench-frozen tissues, it is possible to measure local mass fractions of diffusible as well as of bound elements. The smallest amount of element that can be detected in an ultrathin section is -v I0-19 g under favorable conditions. Since methodology, as stated above, is a major constraint in obtaining accurate information on both the qualitative and quantitative distribution of elements in the specimen, preparatory procedures are emphasized in this volume. Instrumentation is presented in a concise manner. The limited space available does not permit the inclu sion of the applications of x-ray microanalysis to an enormously wide range of bio medical problems. Studies of muscle and isolated cells and liquid droplets are in cluded as examples of its applications. This volume is offered with the hope that it will lead to an understanding of the underlying principles, advantages, and limitations of x-ray microanalysis. Refinements in methodology should follow. It is hoped, further more, that the approach taken in this volume would help the reader to at least under stand when, where, and how artifacts are introduced. This book was written by the joint efforts of ten distinguished scientists and aca demicians, each of whom is an authority in his or her area of specialty. The authors were more than cooperative and prompt, in spite of the fact that they have been very busy carrying on research and teaching. It is a pleasure to acknowledge the fact that I have had the good fortune to collaborate with these scientist-authors, and I have found them extraordinarily competent. M.A. Hayat Chapter 1 PRINCIPLES AND INSTRUMENTATION Alan T. Marshall Zoology Department, La Trobe University, Bundoora, Victoria, Australia GENERATION OF X·RAYS Characteristic X·Rays Inelastic Scattering Continuum Radiation Elastic Scattering INTENSITY OF CHARACTERISTIC X-RAYS Bulk Specimens Thin Sections INTENSITY OF CONTINUUM RADIATION Bulk Specimens Thin Sections PEAK-TO-BACKGROUND RATIOS SPATIAL RESOLUTION ENERGY-DISPERSIVE DETECTOR DETECTOR RESOLUTION DETECTOR EFFICIENCY DETECTOR GEOMETRY Window Thickness Take-off Angle Solid Angle Collimation BACKSCATTERED ELECTRONS WINDOWLESS DETECTORS AMPLIFIER AND PULSE PROCESSOR Pulse Pile-up Rejection Base-Line Restoration Live-Time Correction MULTICHANNEL ANALYZER A LOW-COST SYSTEM MICROSCOPES Available Systems Beam Current Beam Current Stability Measuring Beam Current Correcting Beam Current Beam Diameter Accelerating Voltage Extraneous X-Ray Radiation Vacuum System 2 Marshall QUALITATIVE ANALYSIS Peak Detection Spurious Peaks Elemental Mapping Line Scanning QUANTITATIVE ANALYSIS Practical Problems MANUAL METHODS OF DATA REDUCTION COMPUTER METHODS OF DATA REDUCTION LITERATURE CITED The literature on electron probe x-ray microanalysis is extensive, and there are now a large number of texts that deal with the basic principles (e.g., Andersen, 1967b; Birks, 1969; Hall, 1971; Beaman and Isasi, 1972; Hall et al., 1972; Marshall, 1975a; Reed, 1975; Chandler, 1976a). Consequently this chapter is restricted to x-ray microanalysis as applied to biological specimens and as carried out by means of energy-dispersive x-ray spectrometers attached to electron microscopes. The energy-dispersive x-ray spectrometer is undoubtedly favored for biological work in preference to the wavelength dispersive x-ray spectrometer, largely because of the ease with which it may be mounted on electron microscope columns of all types, its greater x-ray cotlection efficiency, its relative ease of operation, and its lower cost. In this chapter both principles and instrumentation are treated from the point of view of biological x-ray microanalysis and the features that are particularly important for biological analyses are stressed. Analyses of both thin sections and bulk specimens are considered. GENERATION OF X-RAYS When electrons interact with atoms in a sample, x-rays are produced that are characteristic of the elements in the sample. There are, in addition, other interactions that are important when considering x-ray micro analysis. The types of interaction are illustrated in Figure 1 and are sum marized below. Characteristic X-Rays For characteristic x-rays to be produced by electron interaction, the energy of the primary electrons must be greater than a minimum value known as the critical excitation potential. When a primary electron of sufficient energy passes through the electron cloud of a sample atom, it may eject an electron from an inner orbital shell. The primary electron loses energy and experiences a small angular change in direction. This is referred to as inelastic scattering. The conse quence of ejection of an electron from an inner orbital shell is twofold.
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