Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time Analysis
Regular price
57.500 KD
inc. VAT
Couldn't load pickup availability
Table of contents
- Cover
- Contentsxii
- Series Prefacev
- Prefacevii
- Contributorsviii
- Part I: Introduction to Fluorescence Microscopy1
- Chapter One. Fluorescence Microscopy3
- 1.1 Introduction3
- 1.2 Microscope design4
- 1.3 Types of illumination5
- 1.4 Light sources7
- 1.5 Filters9
- 1.6 Objectives and eyepieces12
- References13
- Part II: Optical Probes and Their Applications15
- Chapter Two. Introduction to Fluorescent Probes: Properties, History and Applications17
- 2.1 Introduction17
- 2.2 Nature of fluorescence and properties of fluorescent probes18
- 2.3 Historical developments19
- 2.4 Applications of fluorochromes in histology and microbiology22
- 2.5 Introduction of acridine orange into cell physiology, cytology and cytochemistry23
- 2.6 General applications of fluorescent probes37
- Acknowledgements38
- References38
- Chapter Three. Intracellular Ion Indicators40
- 3.1 Introduction40
- 3.2 General properties of intracellular ion indicators40
- 3.3 Examples of intracellular ion indicators46
- 3.4 Conclusions49
- Acknowledgements49
- References49
- Chapter Four. Fluorescent Imaging of Nucleic Acids and Proteins in Gels51
- 4.1 Introduction51
- 4.2 General properties of fluorescent nucleic acid stains52
- 4.3 Examples of fluorescent nucleic acid gel stains52
- 4.4 General properties of fluorophore labels used to detect nucleic acids55
- 4.5 General properties of fluorescent protein gel stains58
- 4.6 Examples of fluorescent protein gel stains58
- 4.7 Protein labelling61
- 4.8 Conclusions61
- Acknowledgements61
- References61
- Part III: Using Optical Probes in Cells – Practicalities, Problems and Pitfalls63
- Chapter Five. Introducing and Calibrating Fluorescent Probes in Cells and Organelles65
- 5.1 Introduction65
- 5.2 General principles of the loading process66
- 5.3 General principles of the calibration process69
- 5.4 Putting principles into practice70
- Acknowledgements80
- References80
- Chapter Six. Electroporation: A Method for Introduction of Non-permeable Molecular Probes82
- 6.1 Introduction82
- 6.2 Basic concept of electroporation83
- 6.3 Electric field generation and monitoring85
- 6.4 Polarization of the outer membrane86
- 6.5 Electropore formation and resealing87
- 6.6 Transmembrane transport88
- 6.7 Practical considerations of electroporation89
- 6.8 Experimental evidence91
- 6.9 Summary92
- Acknowledgements92
- References93
- Chapter Seven. Imaging Reality: Understanding Maps of Physiological Cell Signals Measured by Fluores94
- 7.1 Introduction94
- 7.2 Generic considerations for the use of fluorescent indicators95
- 7.3 Optimization of fluorescent light detection and background light correction100
- 7.4 3-D spatial maps of fluorescent signals102
- Acknowledgements106
- References106
- Chapter Eight. Fluorescent Probes in Practice – Potential Artifacts108
- 8.1 Introduction108
- 8.2 Photobleaching108
- 8.3 Dynamic range109
- 8.4 Probe loading110
- 8.5 Ion calibration111
- 8.6 Cell movement and fast ion fluxes111
- 8.7 Autofluorescence112
- 8.8 Interactions between multiple probes112
- 8.9 Averaging and intensifier noise113
- 8.10 Probe leakage and exocytosis113
- 8.11 Probe kinetics113
- References113
- Part IV. Optical Probes for Specific Molecules, Organelles and Cells115
- Chapter Nine. Acridine Orange as a Probe for Cell and Molecular Biology117
- 9.1 Introduction117
- 9.2 Historical remarks117
- 9.3 AO as a fluorescent dye118
- 9.4 Spectral properties of AO in complexes with nucleic acids and other biopolymers118
- 9.5 AO in the study of nucleic acids in vitro119
- 9.6 AO in nucleic acid cytochemistry120
- 9.7 AO DNA staining after acid pretreatments122
- 9.8 AO in the study of DNA thermal denaturation122
- 9.9 AO in the study of the chromatin functional state123
- 9.10 Fluorescence polarization of AO in studies of biopolymers126
- 9.11 AO in chromosome banding127
- 9.12 AO in acid polysaccharide histochemistry127
- 9.13 AO binding to proteins128
- 9.14 AO binding to a living cell128
- 9.15 AO in the study of cell viability131
- 9.16 AO in the study of apotopsis131
- 9.17 AO in flow cytometry132
- 9.18 Other applications of AO132
- References132
- Chapter Ten. Fluorescent Lipid Analogues: Applications in Cell and Membrane Biology136
- 10.1 Introduction136
- 10.2 Fluorescent lipid analogues138
- 10.3 Applications143
- Acknowledgements154
- References154
- Chapter Eleven. Optical Probes for Cyclic AMP156
- 11.1 Rationale for creating optical probes for cyclic AMP156
- 11.2 Previous methods for measuring cAMP or imaging related molecules157
- 11.3 Alternative cAMP binding sites158
- 11.4 Properties of A-kinase158
- 11.5 Fluorescent labelling of A-kinase160
- 11.6 Properties of FlCRhR163
- 11.7 Introduction of FlCRhR into cells166
- 11.8 Imaging of FlCRhR and free cAMP167
- 11.9 Applications170
- References171
- Part V: Technology for Qualitative and Quantitative Detection of Optical Probes in Living Cells173
- Chapter Twelve. Quantitative Digital Imaging of Biological Activity in Living Cells with Ion-sensiti175
- 12.1 Introduction175
- 12.2 Fluorescent probes for living cell function176
- 12.3 Observing biological activity in 'real time'178
- 12.4 Ratiometric imaging of ion-sensitive flourescent probes178
- 12.5 Imaging strategies178
- 12.6 Digital image processing181
- 12.7 Data presentation182
- 12.8 Confocal laser scanning microscopy – optical approaches to enhanced image resolution182
- 12.9 A novel high-speed digital confocal microscope183
- 12.10 Digital deconvolution and digital confocal microscopy – 'soft' approaches to enhanced image185
- 12.11 Fluorescent measurements of cytosolic ions – combined photometry with electrophysiology186
- 12.12 Photometric measurements in single cells186
- 12.13 Measurement of fluorescent light – photometry versus imaging187
- 12.14 Photomultiplier tube technology189
- 12.15 Photon counting versus photocurrent integration189
- 12.16 Excitation filter switching190
- 12.17 Dual-emission probes190
- 12.18 Electrophysiology combined with photometry or imaging191
- 12.19 Data acquisition191
- 12.20 CONCORD – integrating imaging, photometry and electrophysiology on a single workstation for191
- 12.21 Cell culture and loading of fluorescent probes193
- 12.22 Calibration of ion-sensitive dyes in living cells and in solution193
- 12.23 Deriving spectra data from optical probes in situ – the SpectralWIZARD194
- 12.24 Summary195
- References195
- Chapter Thirteen. Fast Photometric Measurements of Cell Function Combined with Electrophysiology196
- 13.1 Fluorescent light measurement197
- 13.2 A fluorescence/electrophysiological recording system197
- 13.3 The photomultiplier tube199
- 13.4 Dual-emission dye measurement systems200
- 13.5 Dual-excitation dye measurement systems201
- 13.6 Analogue signal digitization203
- 13.7 Fluorescence measurement systems203
- 13.8 Software for recording fluorescence signals205
- 13.9 The 'chart recorder' paradigm205
- 13.10 The 'oscilloscope' paradigm205
- 13.11 Leak current subtraction206
- 13.12 Computer system designs207
- 13.13 Conclusion208
- References208
- Equipment suppliers208
- Chapter Fourteen. Potentiometric Membrane Dyes and Imaging Membrane Potential in Single Cells210
- 14.1 Introduction210
- 14.2 Optimization of dye indicator sensitivity211
- 14.3 Mapping membrane potential by digital fluorescence microscopy215
- 14.4 Conclusion219
- Acknowledgements219
- References219
- Chapter Fifteen. Fast Multisite Optical Measurement of Membrane Potential, with Two Examples222
- 15.1 Introduction222
- 15.2 Signal type223
- 15.3 Dyes224
- 15.4 Measuring technology225
- 15.5 Two examples229
- 15.6 Population signals from vertebrate brain233
- 15.7 Future directions235
- Acknowledgements236
- References236
- Chapter Sixteen. Imaging Membrane Potential Changes in Individual Neurons238
- 16.1 Introduction238
- 16.2 Extracellular application of voltage-sensitive dyes240
- 16.3 Intracellular application of voltage-sensitive dyes240
- 16.4 Dye injection241
- 16.5 Optical recording241
- 16.6 Multisite recording242
- 16.7 Comparison of optical and electrical signals243
- 16.8 Dye sensitivity (dF/F) and signal-to-noise ratio244
- 16.9 Calibration of the voltage-sensitive dye measurements in terms of membrane potential244
- 16.10 Pharmacological effects and photodynamic damage245
- 16.11 Imaging spike trigger zone246
- 16.12 Vertebrate neurons246
- 16.13 Summary247
- References247
- Part VI: Using Novel Indications for Genetic, Molecular and Cellular Function249
- Chapter Seventeen. Bioluminescent and Chemiluminescent Indicators for Molecular Signalling and Funct251
- 17.1 The natural history of bio- and chemiluminescence251
- 17.2 The analytical potential of chemiluminescent compounds253
- 17.3 Application of chemi- and bioluminescence to living cells256
- 17.4 Bioluminescent reporter genes261
- 17.5 Bioluminescent indicators for molecular signalling in live cells266
- 17.6 Conclusions and future prospects269
- References269
- Chapter Eighteen. Luminescence Imaging of Gene Expression in Single Living Cells273
- 18.1 Introduction273
- 18.2 General considerations274
- 18.3 Equipment required276
- 18.4 Experimental procedures279
- 18.5 Concluding remarks282
- Acknowledgements282
- References282
- Chapter Nineteen. Enhanced Variants of the Green Fluorescent Protein for Greater Sensitivity, Differ284
- 19.1 Introduction284
- 19.2 GFP variants286
- 19.3 Detection of apoptosis with GFP289
- References291
- Chapter Twenty. Rapid Detection of Microorganisms293
- 20.1 Materials and methods296
- 20.2 Results297
- 20.3 Discussion298
- References300
- Part VII: Applications of Confocal Microscopy for Optical Probe Imaging301
- Chapter Twenty-One. Confocal Microscopy – Principles, Practice and Options303
- 21.1 Introduction303
- 21.2 Advantages of scanning303
- 21.3 Confocal microscopy304
- 21.4 Practical aspects306
- 21.5 System performance308
- References309
- Chapter Twenty-Two. Dual-excitation Confocal Fluorescence Microscopy310
- 22.1 Introduction310
- 22.2 Measurement of pHi with BCECF310
- 22.3 Design of a dual-excitation laser scanning confocal fluorescence microscope for measuring pHi w312
- 22.4 Measurement of pHi in the cortical collecting tubule313
- 22.5 Measurement of intracellular Ca 2+ in the perfused afferent arteriole314
- 22.6 Future development315
- Acknowledgements315
- References315
- Chapter Twenty-Three. High-speed Confocal Imaging in Four Dimensions316
- 23.1 Introduction316
- 23.2 Key questions relating to data sampling317
- 23.3 Noran Odyssey XL real-time confocal system317
- 23.4 Spatial and temporal resolutions in 2-D imaging318
- 23.5 Case studies on 2-D resolutions321
- 23.6 Spatial and temporal resolutions in 3-D imaging324
- 23.7 4-D temporal resolution325
- 23.8 3-D and 4-D image processing, display and storage326
- 23.9 Case studies on 3-D and 4-D resolutions327
- 23.10 Future directions329
- Acknowledgements330
- References330
- Chapter Twenty-Four. Multiphoton Fluorescence Microscopy331
- 24.1 Introduction331
- 24.2 Principles of two-photon fluorescence331
- 24.3 The costs and the benefits333
- 24.4 Hardware for two-photon microscopy334
- Acknowledgements335
- References335
- Chapter Twenty-Five. High-resolution Confocal Imaging of Elementary Ca 2+ Signals in Living Cells337
- 25.1 Calcium as an intracellular messenger337
- 25.2 Investigating elementary Ca 2+ signals339
- 25.3 The type of confocal microscope and the mode of scanning341
- 25.4 Additional considerations342
- 25.5 Summary343
- References343
- Chapter Twenty-Six. Confocal Fluorescent Microscopy Using a Nipkow Scanner344
- 26.1 Introduction344
- 26.2 Construction of the CSU10345
- 26.3 Characteristics of the CSU10345
- 26.4 System integration of the CSU10347
- 26.5 Applications347
- 26.6 Future prospects348
- 26.7 Conclusion349
- References349
- Chapter Twenty-Seven. Optimizing Confocal Microscopy for Thick Biological Specimens350
- 27.1 Introduction350
- 27.2 Advantages of confocal over wide-field microscopy, especially for thick biological specimens351
- 27.3 Pathlength errors352
- 27.4 Dye concentration and photobleaching artifacts353
- 27.5 Ratiometric analysis353
- 27.6 Aberrations and confocal microscopy354
- 27.7 Spherical aberration354
- 27.8 Chromatic aberration355
- 27.9 Avoiding refractive index mismatch356
- Acknowledgements360
- References360
- Chapter Twenty-Eight. Redox Confocal Imaging: Intrinsic Fluorescent Probes of Cellular Metabolism361
- 28.1 Introduction361
- 28.2 History of the use of intrinsic probes to monitor cellular metabolism363
- 28.3 The biochemical basis of intrinsic fluorescent probes in living cells364
- 28.4 Instrumentation for the use of low-light-level fluorescent imaging of living cells and tissues365
- 28.5 Applications of intrinsic fluorescent redox probes to cellular metabolism370
- 28.6 Comparison with other non-invasive techniques372
- 28.7 Summary and conclusions373
- Acknowledgements373
- References373
- Part VIII: Advanced Imaging and Light Detection Approaches for Optical Probe Applications375
- Chapter Twenty-Nine. Confocal Raman Microspectrometry377
- 29.1 Introduction377
- 29.2 Raman spectroscopy378
- 29.3 The confocal Raman microspectrometer (CRM)382
- 29.4 Applications385
- 29.5 Future developments402
- Acknowledgements404
- References405
- Chapter Thirty. Spectral Imaging of Autofluorescence Molecules and DNA Probes407
- 30.1 Introduction407
- 30.2 The SpectraCube TM system and design408
- 30.3 The analysis of a spectral image409
- 30.4 Applications of spectral imaging in biology410
- References412
- Chapter Thirty-One. Multiphoton Excitation Microscopy and Spectroscopy of Cells, Tissues and Human S414
- 31.1 Introduction414
- 31.2 History of two-photon excitation microscopy415
- 31.3 Physics of multiphoton excitation processes416
- 31.4 Comparison with confocal microscopy418
- 31.5 Instrumentation for multiphoton excitation microscopy, spectroscopy and lifetime measurement420
- 31.6 Applications to cells and tissues423
- 31.7 Application to in vivo functional imaging of human skin: an example of a thick, highly scatteri425
- 31.8 Mitigation of photodamage with multiphoton excitation microscopy428
- 31.9 Discussion429
- 31.10 Summary and conclusions430
- Acknowledgements430
- References430
- Chapter Thirty-Two. Raman Spectroscopic Methods for In Vitro and In Vivo Tissue Characterization433
- 32.1 Introduction433
- 32.2 Instrumentation434
- 32.3 Calibration of Raman spectra437
- 32.4 Data analysis440
- 32.5 Examples of tissue characterization by Raman spectroscopy443
- 32.6 Conclusion453
- Acknowledgements453
- References454
- Chapter Thirty-Three. In Vivo Semiquantitative NADH-fluorescence Imaging456
- 33.1 Introduction456
- 33.2 Origin of UV-excited (365 nm) tissular fluorescence457
- 33.3 Methodology of semiquantitative in vivo NADH-fluorescence imaging458
- 33.4 Instrumentation460
- 33.5 Experimental verification of the linear relationship between [NADH] and FNADH/Rdiff, 365461
- 33.6 Biomedical and clinical applications of NADH-fluorescence imaging461
- 33.7 Outlook465
- Acknowledgements465
- References465
- Chapter Thirty-Four. Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and Applicatio467
- 34.1 Introduction467
- 34.2 Experimental realizations of lifetime-resolved imaging microscopy468
- 34.3 Implementation of a wide-field frequency-domain FLIM system473
- 34.4 Example of FLIM measurement: GFP in living cells475
- 34.5 Applications of FLIM475
- 34.6 Concluding remarks477
- Acknowledgements478
- References478
- Chapter Thirty-Five. Detection of Flnorescently Labelled Proteins following Gel Electrophoresis: A K480
- 35.1 Introduction480
- 35.2 General application areas for fluorescent imaging in electrophoresis480
- 35.3 What is electrophoresis?481
- 35.4 The use of slab gels482
- 35.5 Visualization of proteins482
- 35.6 Fluorescent prelabelling of proteins482
- 35.7 Imaging fluorescent proteins in gels484
- 35.8 Alternative staining and visualization techniques485
- 35.9 Imaging area485
- 35.10 High-throughput acquisition486
- 35.11 Examples of specific application areas486
- 35.12 Summary487
- References488
- Part IX: CCD Cameras: Key Enabling Technologies for Optical Probe Imaging489
- Chapter Thirty-Six. Properties of Low-light-level Intensified Cameras491
- 36.1 Introduction491
- 36.2 Image intensifier: first and second generation492
- 36.3 Maximizing signal and minimizing noise493
- 36.4 Thermal noise493
- 36.5 Non-thermal noise493
- 36.6 Temporal characteristics of photocathode noise494
- 36.7 Gain use and abuse494
- 36.8 Spectral response495
- 36.9 Comparison between second- and third-generation intensifier tubes496
- 36.10 High-resolution intensifiers497
- 36.11 Fibreoptics or lenses for image transfer?497
- 36.12 The charge-coupled device498
- 36.13 Frame and line transfer CCDs499
- 36.14 Full-frame CCDs499
- 36.15 CCD pixel size and dynamic range500
- 36.16 Read-out features500
- 36.17 High-resolution digital ICCDs500
- 36.18 Fast read-out ICCDs501
- 36.19 Digital ICCDs with increasing bit range502
- 36.20 Analogue video ICCDs502
- 36.21 Photon counting imaging503
- 36.22 Systems integration504
- 36.23 Flat-field normalization- the magic touch506
- Chapter Thirty-Seven. Properties of Low-light-level Slow-scan Detectors507
- 37.1 Introduction507
- 37.2 Contemporary image-acquisition technology507
- 37.3 How CCDs work508
- 37.4 The high-performance slow-scan CCD camera510
- 37.5 Slow-scan CCD camera performance511
- 37.6 Applications of slow-scan CCD cameras514
- 37.7 Summary516
- References516
- Chapter Thirty-Eight. High-speed Digital CCD Cameras- Principles and Applications517
- 38.1 Introduction517
- 38.2 The need for high-speed digital CCD cameras517
- 38.3 Other technological advances519
- 38.4 Systems selection522
- 38.5 Conclusions524
- Part X: Flow Cytometric Methodologies for Measurement of Optical Probes in Live Cells525
- Chapter Thirty-Nine. Flow Cytometry: Use of Multiparameter Kinetics to Evaluate Several Activation P527
- 39.1 Introduction527
- 39.2 Fluorescent techniques – general advantages and disadvantages528
- 39.3 Flow cytometry for kinetic studies of cellular functions529
- 39.4 Time of onset of initial response529
- 39.5 Parameters which can be measured530
- 39.6 Classes of fluorescent probes530
- 39.7 Specific probes for parameters of cell function532
- 39.8 Non-kinetic applications of relevance to multiparameter flow kinetics correlations537
- 39.9 Conclusion538
- Acknowledgements538
- References538
- Chapter Forty. Photolabile Caged Compounds540
- 40.1 Introduction540
- 40.2 Properties of a photolabile probe541
- 40.3 The chemistry of photolabile compounds542
- 40.4 Application of photolabile probes to studying biological pathways546
- 40.5 Potential problems associated with the use of caged compounds548
- 40.6 Conclusion552
- Acknowledgements552
- References552
- Chapter Forty-One. Fluorescent Analogues: Optical Biosensors of the Chemical and Molecular Dynamics554
- 41.1 Introduction554
- 41.2 MeroCaM 1 and 2: fluorescent indicators of calcium-calmodulin binding555
- 41.3 Fluorescent analogue of myosin II557
- 41.4 Protein-based optical biosensors of myosin II regulatory light chain phosphorylation562
- 41.5 Future studies564
- References565
- Part XI: Applications of Optical Probe Imaging to Biological Problems567
- Chapter Forty-Two. Fluorescence and Luminescence Techniques to Probe Ion Activities in Living Plant569
- 42.1 Introduction569
- 42.2 Tissue preparation, mounting and perfusion570
- 42.3 Securing the specimen for microscopy571
- 42.4 Selection and use of fluorescent probes571
- 42.5 Observation and measurement of dye fluorescence578
- 42.6 Calibration in vitro and in situ581
- 42.7 Additional measurement techniques584
- 42.8 Using recombinant aequorin for measurement of intracellular calcium in plants585
- 42.9 Manipulation of intracellular events using caged probes591
- 42.10 Probes for other compartments592
- 42.11 Future developments593
- Acknowledgements594
- References594
- Chapter Forty-Three. Nuclear Calcium: Concepts and Controversies597
- 43.1 Introduction597
- 43.2 The nuclear envelope and nuclear transporters598
- 43.3 Nuclear calcium599
- 43.4 Summary600
- References600
- Chapter Forty-Four. Assessment of Gap Junctional Intercellular Communication in Living Cells Using F602
- 44.1 Introduction602
- 44.2 Materials and methods604
- 44.3 Results606
- 44.4 Discussion609
- Acknowledgements611
- References611
- Chapter Forty-Five. Photoactivation of Fluorescence as a Probe for Cytoskeletal Dynamics in Mitosis613
- 45.1 Introduction613
- 45.2 Photoactivatable fluorescent probes615
- 45.3 A computer-controlled, multiple-channel fluorescence microscope for photoactivation617
- 45.4 Experiments using photoactivation of fluorescence621
- 45.5 Future prospects and conclusions626
- References626
- Note added in proof627
- Chapter Forty-Six. Video Imaging of Lipid Order628
- 46.1 Introduction628
- 46.2 Theory629
- 46.3 Experiment631
- 46.4 Biological applications632
- Acknowledgements633
- References633
- Index634
- Color Plate Section649
The use of fluorescent and luminescent probes to measure biological function has increased dramatically since publication of the First Edition due to their improved speed, safety, and power of analytical approach. This eagerly awaited Second Edition, also edited by Bill Mason, contains 19 new chapters and over two thirds new material, and is a must for all life scientists using optical probes.
The contents include discussion of new optical methodologies for detection of proteins, DNA and other molecules, as well as probes for ions, receptors, cellular components, and gene expression. Emerging and advanced technologies for probe detection such as confocal laser scanning microscopy are also covered. This book will be essential for those embarking on work in the field or using new methods to enhance their research.
TOPICS COVERED:
* Single and multiphoton confocal microscopy
* Applications of green fluorescent protein and chemiluminescent reporters to gene expression studies
* Applications of new optical probes for imaging proteins in gels
* Probes and detection technologies for imaging membrane potential in live cells
* Use of optical probes to detect microorganisms
* Raman and confocal raman microspectroscopy
* Fluorescence lifetime imaging microscopy
* Digital CCD cameras and their application in biological microscopy
The contents include discussion of new optical methodologies for detection of proteins, DNA and other molecules, as well as probes for ions, receptors, cellular components, and gene expression. Emerging and advanced technologies for probe detection such as confocal laser scanning microscopy are also covered. This book will be essential for those embarking on work in the field or using new methods to enhance their research.
TOPICS COVERED:
* Single and multiphoton confocal microscopy
* Applications of green fluorescent protein and chemiluminescent reporters to gene expression studies
* Applications of new optical probes for imaging proteins in gels
* Probes and detection technologies for imaging membrane potential in live cells
* Use of optical probes to detect microorganisms
* Raman and confocal raman microspectroscopy
* Fluorescence lifetime imaging microscopy
* Digital CCD cameras and their application in biological microscopy