Handbook of Microscopy

Handbook of Microscopy is a manual that deals mainly with the basic instruments and techniques used in light microscopy and its biological applications. A large section is devoted to the study of organic matter in microfossils preserved in rocks, in view of its stratigraphic importance in mining and oil prospecting. This text is comprised of six chapters; the first of which introduces the reader to the basic principles as well as to the instruments and techniques used in light microscopy. This book also discusses the microscopes and electronic flashlights for photomicrography, along with the use of monochromatic light, stereological and physicochemical microanalysis, microanalysis by electron microscopy, and microdetermination of physical values. Attention then turns to staining and impregnation and methods of fixation, examination, cutting, and mounting. The remaining chapters focus on the microscopy of topological stains and non-specific cytological stains, with emphasis on special methods used in animal and plant histology and protistology and mycological methods in pathology. This book is written specifically for microscopists.

Preface

Acknowledgments

Abbreviations

1 Instruments and Techniques

1.1 Basic Principles

Light and Colors

Image Formation

Mechanical Conventions

Aberrations

Depth of Field

Flatness of Field

Microscopic Image in Wave Optics

Useful Magnification

Homogeneous Immersion of Objectives

Antireflection Treatment of Optical Surfaces

1.2 Basic Instruments

Mechanical Features of the Microscope

Use of the microscope

Microscopic Observation

1.3 Chief Instrumental Methods

Dark Ground Illumination

Examination under Reflected Light

Infrared Microscopy

Ultraviolet Microscopy

Pupillary Modulators

Interference Microscopes

Phase Contrast

Polarizing Microscope

Study of Anisotropy of Biological Tissues under Polarized Light

Fluorescence Microscopy

Projection Microscopy

1.4 Recording of Images

Photomicrography

Photomacrography

Illuminating during Photomicrography

Colored Filters

Color Differences in Emission and Reception

Black and White Photography

Procedures for Color Photography

Balanced Color Compensation

Variations and Deficiencies in Color Emulsions

Emulsions

Exposure Time

Measurement of the Exposure Time

Development

Printing on Paper

Classification

Transposition of Contrasts

Special Effects in Photomicrography

Equidensitometry

1.5 Synthesis

Stereoscopic Photomicrographs

Graphic Reconstructions

Solid Reconstructions

Cine Reconstructions

Cinemicrography

Drawing at the Microscope

Artifacts

1.6 Measurements

Measuring Techniques

1.7 Special Techniques

Locating Objects

Special Stages and Warmed or Cooled Stages

Microscopic Observations under Increased Pressure

Automation in Microscopy

Microsurgery

1.8 Use of Monochromatic Light

Monochromators

Spectrographs

Spark Microspectroscopes and Emission Spectrography

Microphotometers and Microspectrophotometers

1.9 Stereological Microanalysis

Counting

Morphometry and Stereology

Microquantimeters

1.10 Physicochemical Microanalysis

Micro-Electrophoresis Cell

Thermal Micromethods

Microrefractometry

Autoradiography

Historadiography

Spodography

1.11 Microanalysis by Electron Microscopy

The Scanning Electron Microscope

The Transmission Electron Microscope

Ultra High-Voltage Electron Microscopy

Ultraviolet Excited Electron Microscopy

Proton Microscopy

Ion Microscopy

1.12 Microdetermination of Physical Values

Microdetermination of Osmotic Pressure

Differential Separation Techniques

Ultracentrifugation of Cells

Determination of Dry Weight of Cellular Constituents

Determination of Constituents by Specific Absorption

2 Methods of Fixation, Examination, Cutting and Mounting

2.1 Methods of Preparation

Test or Reference Preparations

Rehydration of Dried Material

Preservation Mixtures

2.2 Methods of Fixation

The Aims of Fixation

Accuracy of Preservation in Fixation

Simple Fixatives

Fixative Mixtures

Time for Fixation

Cytological Fixatives

Embedding Compatibility

Penetration

Compatibility of Stains

Decalcification

Quantity of Fixative

Choice Based on the Specimen

Stopping Fixation

Fixatives for Electron Microscopy

2.3 Methods of Bleaching

Demineralization and Softening

Softening of Keratin and Chitin

Maceration

Depigmentation

2.4 Observations of Living Specimens

Examination of Living Specimens

Vital Staining

Methods of Preparation for Examination of Microorganisms In Vivo

Equilibrated Media

Osmodialysis

2.5 Special Techniques

Thin Sections Produced by Polishing

Disintegration Techniques

Contrast-Enhancing Techniques

Drying and Dehydration

Dehydration in the Liquid Phase

Clearing Agents: Removal of Ethanol

Dehydrating Agents Which Can Be Used Immediately before Paraffin

Clearing Agents Which Can Be Used between 95% Ethanol and Paraffin

2.6 Embedding Methods

Non-Aqueous Media

Aqueous Media

Embedding in Paraffin

Embedding in Celloidin or Collodion

Embedding in Epoxy Resins, Methacrylate and Polyesters

Embedding in Hydrophilic Media

Embedding with Gel-Paraffin Mixtures

2.7 Sections

Microtomes

Cutting Tools

Automatic Honing Equipment

Cutting with a Microtome

Smoothing and Attachment of Sections

Removal of Paraffin before Staining

Ultrafine Sections for Transmission Electron Microscopy

Preparation of Supporting Membranes for Electron Microscopy

Fixation and Preservation of Small Specimens

2.8 Mounting of Preparations

Microscopic preparation

Media for Temporary observation

Permanent Mounting

Visibility Indices in the Mounting Media

Air-tightness of Cements and Varnishes

3 Stains

3.1 Principal Stains

Anthraquinones

Azines

Monoazides

Polyazides

Cyanines, Quinolines

Diazonium, Tetrazonium, Tetrazolium Salts

Indulins

Nitro-Stains

Oxazines

Pyrazolones

Quinone-Oximes

Phthalocyanins

Thiazines

Triarylmethanes

Xanthenes

Thiazoles

Polymethines

Mineral Dyes

Natural Pigments

Fluorochromes

3.2 Synonyms of the Most Important Stains

Glossary of Stains and Dyes

4 Staining and Impregnation

4.1 Methodology

General Considerations

Observations

Staining Materials

Modes of Action

Neutraistains

Staining Whole Pieces

Negative Stains

Infiltration and Injection

Automation of Staining

4.2 Applications

Non-specific Stains

5 Topological Stains

5.1 General Panoptic Methods

Staining of Nuclei with Progressive Haematein Lakes

Trichromes and Dichromes

5.2 Special Methods in Animal Histology

Staining of Blood and Intracellular Parasites

Staining of Bony and Cartilaginous Tissues

Staining of Muscle Fibers

Staining of the Nervous System

Bacteriological Stains

Staining of Tissues Containing Collagen

Staining of Glycogen and Galactogen

Staining of Integuments

Pigments

5.3 Special Methods in Plant Histology

Spores and Cysts

Plant Techniques

Detection of Parasitic Fungi in Plants

Mycological Techniques

5.4 Special Methods in Protistology

Techniques of Protistology

Amoeba

Sporozoa

Flagellates

Infusoria

Spirochaetae

5.5 Mycological Methods in Pathology

Zoopathological Techniques

5.6 Observations on Supplementary Techniques

Automation of Fixation, Impregnation and Staining

Large-Scale Series for Teaching

6 Non-specific Cytological Stains

6.1 General Cytology

6.2 Staining of Intracellular Organelles

6.3 Cellular Diagnosis in Cancer

6.4 Conclusions

Appendix I Physicochemical Constants

Bibliography

Index