MICROSCOPE PHOTOMETRY

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2 Horst Piller MICROSCOPE PHOTOMETRY With 79 Figures Springer-Verlag Berlin Heidelberg New York 1977

3 DR. HORST PILLER C/O Carl Zeiss Postfach 35/36 D-7082 Oberkochen ISBN-13: DOI:IO e-isbn-13: Library of Congress Cataloging in Publication Data. Piller, Horst. Microscope photometry. I. Microphotometry. 1. Title. QH257.P ' 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/

4 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

5 Contents Chapter 1 General Introduction.... Chapter 2 Ray Paths in the Microscope Introduction Illumination Systems for Bright Field Imaging Systems for Bright Field Dark-Field Systems Diaphragms Instrumental Parameters Definition of Geometrical Optical Terms in Alphabetical Order Chapter 3 Transfer of Light Energy in the Optical System Introduction Spectral Concentration of Radiance of the Lamp Spectral Bandwidth Optical Flux Transmittance of the Optical System Interaction of Light with the Stage Object Relation Between Light Power and Photometric Measuring Values Sensation of Brightness in the Observer 37 Chapter 4 The Light Source and Its Power Supply Introduction Stabilised Electric Power Supply Various Types of Lamps The Collector Light Modulators. 49 Chapter 5 Monochromatising Devices and Filters Introduction Characteristics of Monochromatising Devices Monochromatic Interference Filters Prism- and Grating-Monochromators Filters Other Than Monochromatising Polarising Devices

6 VIII Contents Chapter 6 The Basic Microscope Assembly Condensers Reflectors for Reflected-Light Work Types of Microscope Stage Objectives Oculars Body Tubes and Tube Optics. 97 Chapter 7 The Photometer System Photometer Heads Photomultiplier Tubes Electrical Conversion Modules 107 Chapter 8 Adjustment of Equipment Microscope Axis and Stage-Rotation Axis Luminous-Field Diaphragm Condenser-Aperture Diaphragm and the Luminous Surface of Lamps Levelling of the Stage Object in Reflected Light Crossing the Po1ars and Orienting the Cross-Wires Chapter 9 Procedures in Photometry with the Microscope Principle of Measurements Photometer Systems Reference Materials Measuring Procedures Computation of Measuring Results Automation in Measuring and Data Processing 138 Chapter 10 Special Techniques in Microscope Photometry Microscope Fluorometry Photometric Determination of Optical-Path Differences Diffuse Reflection from Surfaces Techniques for Measuring Micro-Autoradiographs Micro-Densitometry Determination of Basic Optical Constants with Interference-Layer Techniques 157 Chapter 11 Special Adjustment for Photometry Focusing of Objective Levelling of Stage Object in Reflected Light Specularly 164

7 Contents IX 11.3 Control of Photometric Field and Luminous Field Control of Angle of Incidence Control of Spectral Bandwidth Setting of Central Wavelength 171 Chapter 12 Systematic Measuring Errors Distinction Between Systematic and Statistical Errors General Aspects of Systematic Errors Instrumental Factors Affecting the Light Supply to the Photosensor Stage-Object Factors Affecting the Light Supply to the Photosensor 185 Chapter 13 Statistical Errors Causes of Statistical Errors Expressions and Terms in Error Statistics Evaluation of Precision with a Limited Number of Tests Comparison of Precision in Different Tests Summation of Standard Deviations 192 Chapter 14 Application of Microscope Photometry Absorption Parameters Measured in Transmitted Light Parameters Measured in Reflected Light Microscope Fluorometry Refraction Parameters Derived from Optical-Path Difference Appendix 1. Specification of a Light Wave Quantities, Units and Symbols Accumulation of Errors Detectivity and the Effect of Statistical Spread Systematic Errors Due to Increments in Measuring Values Experimental Determination of Factors Involved in the Spectral Sensitivity of Measuring Equipment Correlation Between Photometric Values and Phase Differences in Double-Beam Interference Microscopy Theory of Interference-Layer Technique Determination of Relative Difference in Refractive Index of Two Contiguous Media Theory of Secondary Effect of Glare

8 x Contents 11. Mathematical Derivation ofthe Distributional Error Beam Splitting in an Inclined Glass Plate and the Effect of Parasitic Images Derivation of the cos 4 -Law Determination of Angles Between the Microscope Axis and Rays Deflected at, or Traversing, the Stage Object References Subject Index 251

Advanced Electronic Circuits

U. Tietze Ch. Schenk Advanced Electronic Circuits Wi th the Assistance of E. Schmid With 570 Figures Springer-Verlag Berlin Heidelberg New York 1978 Dr.-Ing. Ulrich Tietze Dr.-Ing. Christoph Schenk Universitat