Table Of ContentLight Microscopy in Biology
The Practical Approach Series
SERIES EDITOR
B. D. HAMES
Department of Biochemistry and Molecular Biology
University of Leeds, Leeds LS2 9JT, UK
See also the Practical Approach web site at http://www.oup.co.uk/PAS
* indicates new and forthcoming titles
Affinity Chromatography Cellular Calcium
Affinity Separations Cellular Interactions in
Development
Anaerobic Microbiology
Cellular Neurobiology
Animal Cell Culture
(2nd edition) if Chromatin
Animal Virus Pathogenesis * Chromosome Structural
Analysis
Antibodies I and II
Clinical Immunology
Antibody Engineering
Complement
if Antisense Technology
* Crystallization of Nucleic
Applied Microbial Physiology
Acids and Proteins
Basic Cell Culture (2nd edition)
Behavioural Neuroscience Cytokines (2nd edition)
Bioenergetics The Cytoskeleton
Biological Data Analysis Diagnostic Molecular
Pathology I and II
Biomechanics—Materials
DNA and Protein Sequence
Biomechanics—Structures and
Analysis
Systems
DNA Cloning 1: Core
Biosensors
Techniques (2nd edition)
Carbohydrate Analysis
DNA Cloning 2: Expression
(2nd edition)
Systems (2nd edition)
Cell-Cell Interactions
DNA Cloning 3: Complex
The Cell Cycle
Genomes (2nd edition)
Cell Growth and Apoptosis DNA Cloning 4: Mammalian
* Cell Separation Systems (2nd edition)
if Drosophila (2nd edition) if Immobilized Biomolecules in
Electron Microscopy in Analysis
Biology Immunochemistry 1
Electron Microscopy in Immunochemistry 2
Molecular Biology
Immunocytochemistry
Electrophysiology
if In Situ Hybridization (2nd
Enzyme Assays edition)
Epithelial Cell Culture lodinated Density Gradient
Essential Developmental Media
Biology Essential Molecular Ion Channels
Biology I and I
* Light Microscopy (2nd edition)
* Eukaryotic DNA Replication
Lipid Modification of Proteins
Experimental Neuroanatomy
Lipoprotein Analysis
Extracellular Matrix
Liposomes
Flow Cytometry (2nd edition)
Mammalian Cell
Free Radicals
Biotechnology
Gas Chromatography
Medical Parasitology
Gel Electrophoresis of Nucleic
Medical Virology
Acids (2nd edition)
MHC Volumes 1 and 2
if Gel Electrophoresis of Proteins
if Molecular Genetic Analysis of
(3rd edition)
Populations (2nd edition)
Gene Probes 1 and 2
Molecular Genetics of Yeast
Gene Targeting
Molecular Imaging in
Gene Transcription
Neuroscience
* Genome Mapping
Molecular Neurobiology
Glycobiology
Molecular Plant Pathology
* Growth Factors and Receptors
and II
Haemopoiesis
Molecular Virology
* High Resolution
Monitoring Neuronal Activity
Chromotography
Mutagenicity Testing
Histocompatibility Testing
* Mutation Detection
HIV Volumes 1 and 2
Neural Cell Culture
if HPLC of Macromolecules
Neural Transplantation
(2nd edition)
Neurochemistry (2nd edition)
Human Cytogenetics I and II
(2nd edition) Neuronal Cell Lines
Human Genetic Disease NMR of Biological
Analysis Macromolecules
Non-isotopic Methods in Pulsed Field Gel
Molecular Biology Electrophoresis
Nucleic Acid Hybridisation RNA Processing I and II
Oligonucleotides and if RNA-Protein Interactions
Analogues
Signalling by Inositides
Oligonucleotide Synthesis
Subcellular Fractionation
PCR 1
Signal Transduction
PCR 2
* Transcription Factors (2nd
*PCR 3:PCR In Situ edition)
Hybridization
Tumour Immunobiology
Peptide Antigens
Photosynthesis: Energy
Last 10 published
Transduction
Chromatin 1.4.98
Plant Cell Biology
Drosophila 2/e 19.3.98
Plant Cell Culture (2nd edition) Molecular Genetic Analysis of
Plant Molecular Biology Populations 2/e 19.3.98
Mutation Detection 15.2.98
Plasmids (2nd edition)
Antisense Technology 18.12.97
Platelets
PCR 3:ISH.12.98
Postimplantation Mammalian
Genome Mapping 21.8.98
Embryos
MHC Volume 2 21.8.97
Preparative Centrifugation
MHC Volume 121.8.97
Protein Blotting
Signalling by Inositides
if Protein Expression Vol 1
* Protein Expression Vol 2
In prod
Protein Engineering
HPLC of Mac 2/e
Protein Function (2nd edition) RNA-Protein Interactions
Protein Phosphorylation Growth Factors and Receptors
Cell Sepn
Protein Purification
Applications Light Mic
ISH2/e
Protein Purification Methods
GEP3/e
Protein Sequencing
Immobilized Biomolecules
Protein Structure (2nd edition)
Eukaryotic DNA Replication
Protein Structure Prediction
Chromosone Structural
Protein Targeting Analysis
Proteolytic Enzymes Protein Expression Vol 1
Light Microscopy in
Biology
Second Edition
A Practical Approach
Edited by
ALAN J. LACEY
Department of Biology and Biochemistry, Brunei,
The University of West London, Uxbridge, Middlesex UB8 3PH, UK
OXFORD
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© Oxford University Press 1999
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Preface
Preparing a new edition of an originally successful first edition must always
result in a choice having to be made as to what to leave out to make room for
any new material. Optical microscopy has developed very fast in the last ten
years and yet the same principles of achieving a good image by the interaction
of light with the matter under investigation are still there. Developing tech-
niques in confocal and near-field microscopy have been given chapters as have
calcium ion and pH imaging which reflect something of the enormous develop-
ment of fluorochromes and fluorescent microscopy. Video cameras and video
recorders used to record images for visual examination or to lead on to image
analysis by subsequent processing in digital or other format have all made
large contributions to the development of optical microscopy.
Basic optical microscopy in which aspects of resolution of fine detail and the
requirement of contrast to make that small detail visible has been retained.
The recording of the image by wet chemistry methods is still a routine require-
ment in many projects but the use of video cameras and printers is beginning
to challenge the dark-room photochemical production of still images. The
basic immunohistochemistry and that of differential staining particularly in the
preparation of tissues and chromosomes has been retained while new empha-
sis has been given to such topics as fluoroprobes for calcium and pH imaging
and microinjection of materials into living cells.
The use of optical microscopy techniques such as near-field and nanovid
to push the resolution beyond the traditionally accepted diffraction limit
attempts to bridge the gap between optical and electron microscopy. Surface
details of living cells are very important components in the study of drug
reaction and these together with cells to surface contacts are assisted by the
techniques of reflection-contrast microscopy and evanescent illumination.
The editor wishes to thank all the many colleagues, and particularly Dr
Peter Hobson, for their patience in technical discussions. Mr D. J. Thomson is
gratefully acknowledged for his help in many practical ways. Thanks are due to
the production team of OUP who have been so helpful in preparing this book
over a long period.
Oxford A. J. L.
Jan 1999
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Contents
List of Contributors xix
Abbreviations xxi
1. Basic optical microscopy 1
A. J. Lacey
1. Introduction 1
2. The microscope and its use 1
3. Summary of the process of image formation in the
microscope 3
Lamp colector lens and condensers 3
4. Kohler ilumination 7
5. Resolution in the microscope 10
6. Magnification 14
7. Interim sumary 14
8. Contrast methods 16
Bright-field 16
Dark-field 17
Phase-contrast 23
Diferential interference contrast (DIC) 25
Fluorescence microscopy 26
Provisional summary of contrast techniques 28
Summary of contrast techniques 29
9. Recording the image 29
Microscope/camera atachments 30
Sumary 37
Acknowledgements 43
References 43
2. Introduction to confocal microscopy 45
P. J. Shaw
1. Introduction 45
2. The problem of out-of-focus light 46
The confocal principle: explanation by ray optics 47
