Microscope Photometry

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 auto­ mation 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.

1 General Introduction.- 2 Ray Paths in the Microscope.- 2.1 Introduction.- 2.2 Illumination Systems for Bright Field.- 2.3 Imaging Systems for Bright Field.- 2.4 Dark-Field Systems.- 2.5 Diaphragms.- 2.6 Instrumental Parameters.- 2.7 Definition of Geometrical Optical Terms in Alphabetical Order.- 3 Transfer of Light Energy in the Optical System.- 3.1 Introduction.- 3.2 Spectral Concentration of Radiance of the Lamp.- 3.3 Spectral Bandwidth.- 3.4 Optical Flux.- 3.5 Transmittance of the Optical System.- 3.6 Interaction of Light with the Stage Object.- 3.7 Relation Between Light Power and Photometric Measuring Values.- 3.8 Sensation of Brightness in the Observer.- 4 The Light Source and Its Power Supply.- 4.1 Introduction.- 4.2 Stabilised Electric Power Supply.- 4.3 Various Types of Lamps.- 4.4 The Collector.- 4.5 Light Modulators.- 5 Monochromatising Devices and Filters.- 5.1 Introduction.- 5.2 Characteristics of Monochromatising Devices.- 5.3 Monochromatic Interference Filters.- 5.4 Prism- and Grating-Monochromators.- 5.5 Filters Other Than Monochromatising.- 5.6 Polarising Devices.- 6 The Basic Microscope Assembly.- 6.1 Condensers.- 6.2 Reflectors for Reflected-Light Work.- 6.3 Types of Microscope Stage.- 6.4 Objectives.- 6.5 Oculars.- 6.6 Body Tubes and Tube Optics.- 7 The Photometer System.- 7.1 Photometer Heads.- 7.2 Photomultiplier Tubes.- 7.3 Electrical Conversion Modules.- 8 Adjustment of Equipment.- 8.1 Microscope Axis and Stage-Rotation Axis.- 8.2 Luminous-Field Diaphragm.- 8.3 Condenser-Aperture Diaphragm and the Luminous Surface of Lamps.- 8.4 Levelling of the Stage Object in Reflected Light.- 8.5 Crossing the Polars and Orienting the Cross-Wires.- 9 Procedures in Photometry with the Microscope.- 9.1 Principle of Measurements.- 9.2 Photometer Systems.- 9.3 Reference Materials.- 9.4 Measuring Procedures.- 9.5 Computation of Measuring Results.- 9.6 Automation in Measuring and Data Processing.- 10 Special Techniques in Microscope Photometry.- 10.1 Microscope Fluorometry.- 10.2 Photometric Determination of Optical-Path Differences.- 10.3 Diffuse Reflection from Surfaces.- 10.4 Techniques for Measuring Micro-Auto-radiographs.- 10.5 Micro-Densitometry.- 10.6 Determination of Basic Optical Constants with Interference-Layer Techniques.- 11 Special Adjustment for Photometry.- 11.1 Focusing of Objective.- 11.2 Levelling of Stage Object in Specularly Reflected Light.- 11.3 Control of Photometric Field and Luminous Field.- 11.4 Control of Angle of Incidence.- 11.5 Control of Spectral Bandwidth.- 11.6 Setting of Central Wavelength.- 12 Systematic Measuring Errors.- 12.1 Distinction Between Systematic and Statistical Errors.- 12.2 General Aspects of Systematic Errors.- 12.3 Instrumental Factors Affecting the Light Supply to the Photosensor.- 12.4 Stage-Object Factors Affecting the Light Supply to the Photosensor.- 13 Statistical Errors.- 13.1 Causes of Statistical Errors.- 13.2 Expressions and Terms in Error Statistics.- 13.3 Evaluation of Precision with a Limited Number of Tests.- 13.4 Comparison of Precision in Different Tests.- 13.5 Summation of Standard Deviations.- 14 Application of Microscope Photometry.- 14.1 Absorption Parameters Measured in Transmitted Light.- 14.2 Parameters Measured in Reflected Light.- 14.3 Microscope Fluorometry.- 14.4 Refraction Parameters Derived from Optical-Path Difference.- Appendix 1. Specification of a Light Wave.- 2. Quantities, Units and Symbols.- 3. Accumulation of Errors.- 4. Detectivity and the Effect of Statistical Spread.- 5. Systematic Errors Due to Increments in Measuring Values.- 6. Experimental Determination of Factors Involved in the Spectral Sensitivity of Measuring Equipment.- 7. Correlation Between Photometric Values and Phase Differences in Double-Beam Interference Microscopy.- 8. Theory of Interference-Layer Technique.- 9. Determination of Relative Difference in Refractive Index of Two Contiguous Media.- 10. Theory of Secondary Effect of Glare.- 11. Mathematical Derivation of the Distributional Error.- 12. Beam Splitting in an Inclined Glass Plate and the Effect of Parasitic Images.- 14. Determination of Angles Between the Microscope Axis and Rays Deflected at, or Traversing, the Stage Object.- References.