Horst Piller MICROSCOPE PHOTOMETRY With 79 Figures Springer-Verlag Berlin Heidelberg New York 1977
DR. HORST PILLER C/O Carl Zeiss Postfach 35/36 D-7082 Oberkochen ISBN-13: 978-3-642-66572-1 DOI:IO.10071 978-3-642-66570-7 e-isbn-13: 978-3-642-66570-7 Library of Congress Cataloging in Publication Data. Piller, Horst. Microscope photometry. I. Microphotometry. 1. Title. QH257.P54. 578'.4. 76-58893. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. by Springer-Verlag Berlin Heidelberg 1977 Softcover reprint of the hardcover 1st edition 1977 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. 2132/3130-543210
Preface Over the last dozen years there has been a great development in the study of matter on the micro-scale by means of the light microscope, which has coincided with the rapid growth of automation and the use of computers. The manufacturers have responded to this challenge, and there is now available a large choice of modules for use with the microscope, both on the biological and non-biological side. This book is on microscope photometry. Photometry on the macro-scale can be applied to features of not less than about one millimetre in size. Below this lies the realm of microscope photometry which will be discussed both in theory and in practice. As a general rule no discussion of technical design of equipment is included, as such information is available in the handbooks of the particular manufacturers of equipment. It attempts to cover all the uses of the optical microscope photometer with its auxiliary equipment, and the first chapter describes the topics covered. In using the word' light' we apply its meaning to extend from UV to near IR. No attempt has been made to list all the works consulted. For each subject treated in this book a list is given of books and papers considered to be of key importance; in these will be found more detailed bibliographies. It is from Carl Zeiss (Oberkochen, West Germany) that this book has sprung; the author would like to express his personal gratitude to Dr. Michel, Dr. Weber and Mr. Muchel. In the work of publication the author is particularly indebted to Dr. K.F. Springer and the staff of Springer-Verlag. Dr. N.F.M. Henry of the Department of Mineralogy and Petrology, University of Cambridge has freely given advice and help with the English text. Oberkochen, April 1977 HORST PILLER
Contents Chapter 1 General Introduction.... Chapter 2 Ray Paths in the Microscope 4 2.1 Introduction... 4 2.2 Illumination Systems for Bright Field 10 2.3 Imaging Systems for Bright Field 12 2.4 Dark-Field Systems.. 13 2.5 Diaphragms...... 14 2.6 Instrumental Parameters. 15 2.7 Definition of Geometrical Optical Terms in Alphabetical Order......... 17 Chapter 3 Transfer of Light Energy in the Optical System 22 3.1 Introduction 22 3.2 Spectral Concentration of Radiance of the Lamp 24 3.3 Spectral Bandwidth 24 3.4 Optical Flux 25 3.5 Transmittance of the Optical System. 33 3.6 Interaction of Light with the Stage Object 35 3.7 Relation Between Light Power and Photometric Measuring Values. 36 3.8 Sensation of Brightness in the Observer 37 Chapter 4 The Light Source and Its Power Supply 41 4.1 Introduction 41 4.2 Stabilised Electric Power Supply. 41 4.3 Various Types of Lamps. 43 4.4 The Collector. 48 4.5 Light Modulators. 49 Chapter 5 Monochromatising Devices and Filters. 51 5.1 Introduction... 51 5.2 Characteristics of Monochromatising Devices.............. 52 5.3 Monochromatic Interference Filters. 57 5.4 Prism- and Grating-Monochromators 64 5.5 Filters Other Than Monochromatising 72 5.6 Polarising Devices.......... 73
VIII Contents Chapter 6 The Basic Microscope Assembly..... 78 6.1 Condensers... 78 6.2 Reflectors for Reflected-Light Work. 80 6.3 Types of Microscope Stage. 85 6.4 Objectives......... 86 6.5 Oculars.......... 94 6.6 Body Tubes and Tube Optics. 97 Chapter 7 The Photometer System.. 99 7.1 Photometer Heads 99 7.2 Photomultiplier Tubes 101 7.3 Electrical Conversion Modules 107 Chapter 8 Adjustment of Equipment..... 112 8.1 Microscope Axis and Stage-Rotation Axis 112 8.2 Luminous-Field Diaphragm.. 112 8.3 Condenser-Aperture Diaphragm and the Luminous Surface of Lamps...... 113 8.4 Levelling of the Stage Object in Reflected Light.......... 116 8.5 Crossing the Po1ars and Orienting the Cross-Wires....... 117 Chapter 9 Procedures in Photometry with the Microscope 120 9.1 Principle of Measurements 120 9.2 Photometer Systems. 121 9.3 Reference Materials... 125 9.4 Measuring Procedures.. 128 9.5 Computation of Measuring Results 131 9.6 Automation in Measuring and Data Processing 138 Chapter 10 Special Techniques in Microscope Photometry. 141 10.1 Microscope Fluorometry....... 141 10.2 Photometric Determination of Optical-Path Differences.............. 143 10.3 Diffuse Reflection from Surfaces.... 149 10.4 Techniques for Measuring Micro-Autoradiographs............. 150 10.5 Micro-Densitometry.......... 154 10.6 Determination of Basic Optical Constants with Interference-Layer Techniques 157 Chapter 11 Special Adjustment for Photometry. 11.1 Focusing of Objective.... 11.2 Levelling of Stage Object in Reflected Light....... 161 161 Specularly 164
Contents IX 11.3 Control of Photometric Field and Luminous Field........... 165 11.4 Control of Angle of Incidence 167 11.5 Control of Spectral Bandwidth 170 11.6 Setting of Central Wavelength 171 Chapter 12 Systematic Measuring Errors.... 172 12.1 Distinction Between Systematic and Statistical Errors............. 172 12.2 General Aspects of Systematic Errors.. 173 12.3 Instrumental Factors Affecting the Light Supply to the Photosensor.... 174 12.4 Stage-Object Factors Affecting the Light Supply to the Photosensor 185 Chapter 13 Statistical Errors....... 189 13.1 Causes of Statistical Errors. 189 13.2 Expressions and Terms in Error Statistics 189 13.3 Evaluation of Precision with a Limited Number of Tests........... 190 13.4 Comparison of Precision in Different Tests 191 13.5 Summation of Standard Deviations 192 Chapter 14 Application of Microscope Photometry.. 194 14.1 Absorption Parameters Measured in Transmitted Light............. 194 14.2 Parameters Measured in Reflected Light 197 14.3 Microscope Fluorometry... 199 14.4 Refraction Parameters Derived from Optical-Path Difference.... 200 Appendix 1. Specification of a Light Wave. 201 2. Quantities, Units and Symbols 202 3. Accumulation of Errors.... 213 4. Detectivity and the Effect of Statistical Spread 214 5. Systematic Errors Due to Increments in Measuring Values............ 217 6. Experimental Determination of Factors Involved in the Spectral Sensitivity of Measuring Equipment............... 220 7. Correlation Between Photometric Values and Phase Differences in Double-Beam Interference Microscopy........... 224 8. Theory of Interference-Layer Technique.. 227 9. Determination of Relative Difference in Refractive Index of Two Contiguous Media.. 230 10. Theory of Secondary Effect of Glare... 230
x Contents 11. Mathematical Derivation ofthe Distributional Error... 233 12. Beam Splitting in an Inclined Glass Plate and the Effect of Parasitic Images....... 235 13. Derivation of the cos 4 -Law........ 238 14. Determination of Angles Between the Microscope Axis and Rays Deflected at, or Traversing, the Stage Object......... 239 References.. 244 Subject Index 251