Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics

462 Pages·1994·20.37 MB·English

by  Theodore George Rochow

Introduction to
Microscopy by Means
of Light, Electrons,
X Rays, or Acoustics
SECOND EDITION
Acoustic micrograph of a composite of boron and glass fibers in an
epoxy matrix. Boron fibers are seen in cross-sectional view and the
smaller glass fibers are in both the longitudinal and cross-sectional
view. The photomicrograph was taken with 400-megahertz acoustic
radiation. (Courtesy of D. A. Downs, A. El-Shiekh, M. H. Mohamed,
P. A. Tucker, and J. C. Russ of North Carolina State University.) (See
Chapter 18.)
Introduction to
Microscopy by Means
of Light, Electrons,
X Rays, or Acoustics
SECOND EDITION
Theodore George Rochow
and
Paul Arthur Tucker
North Carolina State University at Raleigh
Raleigh, North Carolina
SPRINGER SCIENCE+BUSINESS MEDIA, LLC
L1brarv of Congress Catalog1ng-1n-Publ1cat1on Data
Rochow, Theodore George.
Introduct1on ta m1croscopy by means of 11ght, electrons, X rays,
ar acoust1cs 1 Theodore George Rochow and Paul Arthur Tucker. -- 2nd
ed.
p. cm.
Rev. ed. of: An 1ntroduct1on ta m1croscopy by means of 11ght,
electrons, X rays, ar ultrasound. c1978.
Includes b1b11ograph1cal references and 1ndex.
ISBN 978-1-4899-1515-3 ISBN 978-1-4899-1513-9 (eBook)
DOI 10.1007/978-1-4899-1513-9
1. M1croscopy. I. Tucker, Paul Arthur. II. Rochow, Theodore
George. Introduct1on to m1croscopy by means of 11ght, electrons, X
rays, ar ultrasound. III. T1tle.
[DNLM· 1. M1croscopy. 2. X Rays. 3. Acoust1cs. OH 205.2 R6811
1994]
OH205.2.R63 1994
502 · . 8 · 2--dc20
DNLM/DLC
for L1brary of Congress 94-15345
CIP
ISBN 978-1-4899-1515-3
© 1994, 1978 Springer Science+Business Media New York
Originally published by Plenurn Press, New York in 1994
Softcover reprint ofthe hardcover 2nd edition 1994
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any
form or by any means, electronic, mechanical, photocopying, microfilming, recording, or
otherwise, without written permission from the Publisher
To the North Carolina
State University at Raleigh
Preface
Following three printings of the First Edition (1978), the publisher has
asked for a Second Edition to bring the contents up to date. In doing so the
authors aim to show how the newer microscopies are related to the older
types with respect to theoretical resolving power (what you pay for) and
resolution (what you get).
The book is an introduction to students, technicians, technologists,
and scientists in biology, medicine, science, and engineering. It should be
useful in academic and industrial research, consulting, and forensics; how
ever, the book is not intended to be encyclopedic.
The authors are greatly indebted to the College of Textiles of North
Carolina State University at Raleigh for support from the administration
there for typing, word processing, stationery, mailing, drafting diagrams,
and general assistance. We personally thank Joann Fish for word process
ing, Teresa M. Langley and Grace Parnell for typing services, Mark Bowen
for drawing graphs and diagrams, Chuck Gardner for photographic ser
vices, Deepak Bhattavahalli for his work with the proofs, and all the other
people who have given us their assistance.
The authors wish to acknowledge the many valuable suggestions
given by Eugene G. Rochow and the significant editorial contributions
made by Elizabeth Cook Rochow.
Theodore G. Rochow
Paul A. Tucker
Raleigh, North Carolina
vii
Contents
1. A Brief History of Microscopy
1.1. Introduction............................................ 1
1.2. Correcting for Aberrations............................... 6
1.3. Condensers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4. Dark-Field Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.5. Polarized Light Microscopy.............................. 9
1.6. Near-Field Scanning Light Microscopes . . . . . . . . . . . . . . . . . . . 11
1.7. Light Microscope Manufacturers.......................... 11
1.8. Transmission Electron Microscopes....................... 12
1.9. Scanning Electron Microscopes........................... 15
1.10. Electron-Probe Microanalyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.11. Field-Emission Microscopes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.12. Scanning Tunneling Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.13. Scanning Acoustic Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.14. Atomic Force Microscopes............................... 18
1.15. X-Ray Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.16. X-Ray Laser Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.17. Microscopy Society of America........................... 19
1.18. Summary.............................................. 20
2. Definitions, Attributes of Visibility, and General Principles
2.1. Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.2. Attributes of Visibility.. . .. . .. .. .. . .. .. . .. . .. .. . . . .. . .. . . 25
2.2.1. Correcting for Aberrations........................ 25
2.2.2. Sample Quantity and Quality . . . . . . . . . . . . . . . . . . . . . 25
2.2.3. Focus Depth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2.4. Focus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2.5. Illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2.6. Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
ix
X Contents
2.2.7. Anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.8. Magnification.................................... 31
2.2.9. Stereoscopy..................................... 32
2.3. General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.1. Specimen Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.2. Specimen Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.3. Specimen Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.4. Experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.5. Specimen Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.6. Specimen Behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.7. Photomicrography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.3.8. Video. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4. Summary............................................... 35
3. Simple and Compound Light Microscopes
3.1. Limits of Resolution by the Eye........................... 37
3.2. Simple Microscopes: One-Lens Systems.................... 38
3.3. Compound Microscopes: Two or More Lens Systems . . . . . . . 40
3.4. Stereocompound Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.4.1. Illuminating with Stereomicroscopes............... 48
3.4.2. Preparing the Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5. Biological Microscopes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5.1. Objectives....................................... 50
3.5.2. Eyepieces....................................... 51
3.5.3. Condensers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.5.4. Illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.5.5. Ultraviolet and Infrared Light . . . . . . . . . . . . . . . . . . . . . 58
3.5.6. Anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.5.7. Magnification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.5.8. Photomicrography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.6. Summary............................................... 59
4. Compound Microscopes Using Reflected Light
4.1. Studying Surfaces by Reflected Light...................... 61
4.2. Resolving Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.3. Contrast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.4. Correcting for Aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.5. Specimen Cleanliness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.6. Focus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.7. Illumination............................................ 68
4.8. Radiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.9. Magnification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.10. Field of View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Contents xi
4.11 Glare ................................................. . 78
4.12. Depth ................................................ . 79
4.13. Working Distance ...................................... . 80
4.14. Structure .............................................. . 81
4.15. Morphology ........................................... . 82
4.16. Information about the Specimen ......................... . 83
4.17. Experimentation ....................................... . 83
4.18. Specimen Behavior ..................................... . 83
4.19. Specimen Preparation .................................. . 86
4.20. Photomicrographic Techniques .......................... . 87
4.21. Summary ............................................. . 87
5. Microscopy with Polarized Light
5.1. Overhead Projections.................................... 89
5.2. Anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
5.3. Numerical Aperture and Interference Figures . . . . . . . . . . . . . . 94
5.4. Resolution: Specimen Interaction and Polarized Light . . . . . . . 95
5.5. Contrast: Michel-Levy Interference Chart. . . . . . . . . . . . . . . . . . 97
5.5.1. Personal Interpretation of Interference Colors....... 99
5.5.2. Retardation Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.5.3. Specimen Thickness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.6. Correcting for Aberrations Due to Strain . . . . . . . . . . . . . . . . . . 101
5.7. Cleanliness: Freedom from Interference Films.............. 102
5.8. Focus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.9. Illumination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.10. Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.11. Magnification.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.12. Field of View of an Interference Figure.................... 105
5.13. Glare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.14. Depth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.15. Working Distance....................................... 106
5.16. Specimen Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.17. Specimen Morphology................................... 107
5.18. Information about the Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.19. Experimentation........................................ 107
5.20. Specimen Behavior. . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . .. . . . 108
5.21. Specimen Preparation................................... 108
5.22. Photography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.23. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
6. Microscopical Properties of Fibers
6.1. Introduction............................................ 113
6.2. Fiber Morphology.. .. . .. .. .. . .. .. .. . .. . .. .. .. .. .. . .. .. .. 113