Light microscopy in biology: a practical approach
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Oxford [u.a.]
Oxford Univ. Press
1999
|
| Ausgabe: | 2. ed. |
| Schriftenreihe: | The practical approach series
195 |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XXII, 452 S. Ill., graph. Darst. |
| ISBN: | 0199636699 0199636702 |
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Datensatz im Suchindex
| _version_ | 1804126990148567040 |
|---|---|
| adam_text | 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
UNIVERSITY PRESS
Contents
List of Con trib u tors xix
Abbreviations xxi
1 Basic opt ical mic roscopy 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 collector lens and condensers 3
4 Kohler illumination 7
5 Resolution in the microscope 10
6 Magnification 14
7 Interim summary 14
8 Contrast methods 16
Bright-field 16
Dark-field 17
Phase-contrast 23
Differential 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 attachments 30
Summary 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
Contents
Linear, shift-invariant imaging and the point spread function 48
The shape of the point spread function 49
Aberrations and the limits to linear, shift-invariant imaging 51
3 Practical implementation of confocal scanning systems 54
Point scanning 54
Slit scanning 55
Spinning disc 56
Two-photon imaging 57
4 Comparison of conventional, wide-field fluorescence
imaging with confocal fluorescence imaging 58
Noise and resolution 58
Out-of-focus light 60
When should confocal microscopy be used? 61
5 Practical examples of specimen preparation for confocal
imaging 63
References 70
3 Video microscopy 73
Dieter G Weiss, Willi Maile, Robert A Wick, and Walter Steffen
1 Video microscopy and the equipment required 73
Introduction 73
General strategies of electronic image improvement 77
The different video-microscopic techniques 78
Electronic equipment for video microscopy 89
Considerations on the microscope 101
2 High resolution: video-enhanced contrast microscopy 106
Different types of video-enhanced contrast microscopy 106
Sample preparation 108
Procedure for image generation 110
Interpretation 116
Typical applications and limitations 117
3 Low light: video-intensified microscopy 123
Introduction 123
Procedure for image generation 123
Typical applications 127
4 Image analysis: video-based techniques for measurements in
living cells 130
Spatial measurements and motion analysis 131
Intensity measurements 132
Contents
5 Documentation and presentation of video microscopy data 133
Video recording 133
Obtaining printouts for presentation and publication 141
Preparing and presenting video sequences 143
Acknowledgements 146
References 146
Further reading 148
4 Microscopy of chromosomes 151
A T Sumner and A R Leitch
1 Introduction 151
2 Methods of preparing chromosomes 151
Routine preparation of mammalian chromosomes 152
Preparation of cells by cytocentrifugation for immunocytochemical
studies of chromosomes 155
Preparation of chromosomes from plant cells 156
Assessment of the quality of chromosome preparations 159
3 Uniform (solid) staining of chromosomes 160
4 Chromosome banding 161
The classification of chromosome bands 161
C-banding 162
G-banding 164
Ag-NOR staining for nucleolus organizing regions 164
CREST labelling of kinetochores 166
5 In situ hybridization 168
Probe preparation 170
In situ hybridization reaction 173
6 Observation and recording of images of chromosomes 177
Observation of banding with absorbing dyes 178
Observation of fluorescent chromosomes 178
Photography of chromosomes 180
Other methods of image capture 181
References 183
5 Immunohistochemistry 185
Michael G Ormerod and Susanne F Imrie
1 Introduction 185
2 Antibodies 186
xi
Contents
Immunoglobulin structure 186
Polyclonal antisera 187
Monoclonal antibodies 187
Purification of antibodies 188
Specificity of antibody reactions 188
Storage of antibodies 189
3 Effect of tissue processing on antigens 189
Choosing conditions for processing 189
Revealing hidden antigens 192
4 Choice of label 194
Fluorescent labels 194
Enzymatic labels 194
Colloidal gold 197
Selecting a label 197
5 Methods of application 197
The direct method 198
The indirect method 198
Enzyme-anti-enzyme methods 199
Systems using biotin-avidin 200
Other methods 201
6 Experimental methods 202
A general method 202
Choosing the correct dilution of antibody 203
Fluorescent labels 204
Peroxidase 204
Alkaline phosphatase 207
Glucose oxidase 209
Galactosidase 209
Immunogold • 209
Some general procedures 210
7 Controls and problem solving 213
Controls 213
Problem solving 213
8 Detecting two antigens on the same section 214
9 Cytological preparations 215
10 Quantification 218
11 Equipment 218
References 219
6 Calcium and pH imaging in living cells
Richard M Parton and Nick D Read
1 Introduction 221
xii
Contents
2 Study of calcium and pH in living cells 221
3 Imaging intracellular free calcium and pH 222
4 Fluorescent dyes for free calcium and pH 223
Properties of calcium and pH dyes 223
Intracellular dye behaviour 225
Single wavelength dyes, ratiometric dyes, and ratiometric dye-pairs 230
5 Introducing calcium and pH dyes into living cells 231
General considerations 231
Ester loading 232
Low pH loading 235
Scrape loading 235
Electroporation 235
Ionophoretic microinjection 236
Pressure microinjection 236
Quantifying the extent of dye loading 237
6 Equipment for fluorescence microscopy 237
Fluorescence microscopes - 237
Objectives 238
Dye excitation sources for ion imaging 239
Filters for ion imaging 239
7 Fluorescence imaging systems 240
General requirements 240
Conventional fluorescence imaging 241
Confocal imaging 243
Multiphoton imaging 244
Imaging with multiple detectors 245
8 Optimizing the performance of imaging systems 245
9 Handling experimental material on the microscope stage 248
10 Digital image processing 249
11 Ratioing 249
12 Quantitative ion imaging 252
Image quality and quantitative imaging 252
Signal-to-noise ratio - 254
Numerical data extraction 256
Quantitative ratio imaging 257
Calibration of dye response 260
Statistical analysis of image data 264
13 Visual presentation of image data 267
Visual image enhancement 267
Preparation of digitized figures and plates for publication 267
14 Combining ion imaging with other experimental techniques 269
15 Critical controls for intracellular ion imaging 270
xiii
Contents
Acknowledgements
References
Reflection-contrast microscopy
J S Ploem, F A Prins, and I Cornelese-ten Velde
1 Introduction
Methodology
Applications
Review articles
2 Optical systems for RCM
Early developments in reftected-light microscopy
New developments in reflection-contrast microscopy
Modern reflection-contrast microscopes
3 Image formation
General
Image formation in RCM of living unstained cells
Image formation in RCM of stained specimens
4 Applications
General
Special applications
5 Specimen preparation
General
Immunohistochemistry
Fixation and embedding
Sectioning
Immuno- and histochemical staining for RCM
Mounting and examining sections on microscope slides
6 Summary
Acknowledgements
References
8 Histomorphometry 311
A J Reynolds
1 Introduction 311
2 Microscopy 313
Specimen preparation 313
Obtaining an image 318
Calibration 319
3 Linear measurements 321
Intercept measurement 321
Point counting 324
xiv
Contents
4 Automated measurement 327
Measurement with an interactive computer system (digitizer tablet) 327
Semi-automatic image analysers 329
5 Histomorphometry 337
Acknowledgements 338
Instrumentation and sources of supply 338
References 339
9 Near-field optical microscopy 341
Niek F van Hulst
1 Introduction
Optical microscopy
Probe microscopy
Breaking the diffraction limit
Scope of this chapter
2 Instrumentation
Probes and distance regulation
Antenna-type or appertureless near-field scanning optical
microscopy
Aperture-type near-field scanning optical microscopy
Photon scanning tunnelling microscopy (PSTM)
Distance regulation: shear force microscopy
Conclusion
3 Applications
Single fluorophores and proteins
Monolayers and aggregates
Virus
Cellular surface and cytoskeleton
Chromosomes and fluorescence in situ hybridization
4 Conclusions
5 Future outlook
Acknowledgements
References
- 344
10 Introduction of materials into living cells 373
1 Particle bombardment as a means of DNA transfer into
plant cells 373
Christian Schopke and Claude M Fauquet
Introduction 373
Practical considerations 374
Identification of cells transformed with reporter genes 380
xv
Contents
2 Microinjection as a preparative technique for microscopical
analysis 386
H F Paterson
Advantages of microinjection by glass capillary needle 386
Equipment required for microinjection of adherent mammalian
cells 387
Preparation of materials for microinjection 391
Microinjection technique 392
Microscopical analysis of microinjected cells 394
Acknowledgements 396
References 396
11 Surface fluorescence microscopy with
evanescent illumination 399
D Axelrod
1 Fluorescence at surfaces 399
TIRF for biochemical samples 399
TIRF for biological samples 401
2 Theory of TIRF 401
Single interfaces: intensity and polarization 401
Intermediate dielectric layers 405
Intermediate metal film 406
3 Optical configurations 407
Inverted microscope TIR with prism on top 407
Inverted microscope TIR with prism below 410
Upright microscope TIR with prism below 411
Inverted microscope TIR without a prism 414
Rapid chopping between TIR and epi-illumination 417
4 General experimental suggestions 417
5 Applications of TIRF microscopy 420
6 Comparison with other optical sectioning microscopies 422
Acknowledgements 423
References 423
12 Nanovid microscopy 425
Greta M Lee
1 Introduction 425
Use of nanovid microscopy to analyse molecular mobility in
membranes 428
Use of nanovid microscopy to study the extracellular matrix 428
xvi
Contents
2 General information on equipment and methods
Microscope
Camera
Image processor and frame grabber
Image storage
3 Image analysis to analyse molecular mobility (single
particle tracking)
References
Appendix I
Appendix II
Index
XVll
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| spelling | Light microscopy in biology a practical approach ed. by Alan J. Lacey 2. ed. Oxford [u.a.] Oxford Univ. Press 1999 XXII, 452 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series 195 IMUNOHISTOQUÍMICA MICROSCOPIA (MÉTODOS;TÉCNICAS) Microscopie - Technique Microscopy Technique Microscopy methods Mikroskopie (DE-588)4039238-7 gnd rswk-swf Biologie (DE-588)4006851-1 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Mikroskopie (DE-588)4039238-7 s DE-604 Biologie (DE-588)4006851-1 s Methode (DE-588)4038971-6 s Lacey, Alan J. Sonstige oth The practical approach series 195 (DE-604)BV007921321 195 HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008382562&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Light microscopy in biology a practical approach The practical approach series IMUNOHISTOQUÍMICA MICROSCOPIA (MÉTODOS;TÉCNICAS) Microscopie - Technique Microscopy Technique Microscopy methods Mikroskopie (DE-588)4039238-7 gnd Biologie (DE-588)4006851-1 gnd Methode (DE-588)4038971-6 gnd |
| subject_GND | (DE-588)4039238-7 (DE-588)4006851-1 (DE-588)4038971-6 |
| title | Light microscopy in biology a practical approach |
| title_auth | Light microscopy in biology a practical approach |
| title_exact_search | Light microscopy in biology a practical approach |
| title_full | Light microscopy in biology a practical approach ed. by Alan J. Lacey |
| title_fullStr | Light microscopy in biology a practical approach ed. by Alan J. Lacey |
| title_full_unstemmed | Light microscopy in biology a practical approach ed. by Alan J. Lacey |
| title_short | Light microscopy in biology |
| title_sort | light microscopy in biology a practical approach |
| title_sub | a practical approach |
| topic | IMUNOHISTOQUÍMICA MICROSCOPIA (MÉTODOS;TÉCNICAS) Microscopie - Technique Microscopy Technique Microscopy methods Mikroskopie (DE-588)4039238-7 gnd Biologie (DE-588)4006851-1 gnd Methode (DE-588)4038971-6 gnd |
| topic_facet | IMUNOHISTOQUÍMICA MICROSCOPIA (MÉTODOS;TÉCNICAS) Microscopie - Technique Microscopy Technique Microscopy methods Mikroskopie Biologie Methode |
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