Fluorescent and Luminescent Probes for Biological Activity (eBook)

Front Cover 1
Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time Analysis 4
Copyright Page 5
Contents 13
Series Preface 6
Preface 8
Contributors 9
Part I: Introduction to Fluorescence Microscopy 28
Chapter One. Fluorescence Microscopy 30
1.1 Introduction 30
1.2 Microscope design 31
1.3 Types of illumination 32
1.4 Light sources 34
1.5 Filters 36
1.6 Objectives and eyepieces 39
References 40
Part II: Optical Probes and Their Applications 42
Chapter Two. Introduction to Fluorescent Probes: Properties, History and Applications 44
2.1 Introduction 44
2.2 Nature of fluorescence and properties of fluorescent probes 45
2.3 Historical developments 46
2.4 Applications of fluorochromes in histology and microbiology 49
2.5 Introduction of acridine orange into cell physiology, cytology and cytochemistry 50
2.6 General applications of fluorescent probes 64
Acknowledgements 65
References 65
Chapter Three. Intracellular Ion Indicators 67
3.1 Introduction 67
3.2 General properties of intracellular ion indicators 67
3.3 Examples of intracellular ion indicators 73
3.4 Conclusions 76
Acknowledgements 76
References 76
Chapter Four. Fluorescent Imaging of Nucleic Acids and Proteins in Gels 78
4.1 Introduction 78
4.2 General properties of fluorescent nucleic acid stains 79
4.3 Examples of fluorescent nucleic acid gel stains 79
4.4 General properties of fluorophore labels used to detect nucleic acids 82
4.5 General properties of fluorescent protein gel stains 85
4.6 Examples of fluorescent protein gel stains 85
4.7 Protein labelling 88
4.8 Conclusions 88
Acknowledgements 88
References 88
Part III: Using Optical Probes in Cells – Practicalities, Problems and Pitfalls 90
Chapter Five. Introducing and Calibrating Fluorescent Probes in Cells and Organelles 92
5.1 Introduction 92
5.2 General principles of the loading process 93
5.3 General principles of the calibration process 96
5.4 Putting principles into practice 97
Acknowledgements 107
References 107
Chapter Six. Electroporation: A Method for Introduction of Non-permeable Molecular Probes 109
6.1 Introduction 109
6.2 Basic concept of electroporation 110
6.3 Electric field generation and monitoring 112
6.4 Polarization of the outer membrane 113
6.5 Electropore formation and resealing 114
6.6 Transmembrane transport 115
6.7 Practical considerations of electroporation 116
6.8 Experimental evidence 118
6.9 Summary 119
Acknowledgements 119
References 120
Chapter Seven. Imaging Reality: Understanding Maps of Physiological Cell Signals Measured by Fluorescence Microscopy and Digital Imaging 121
7.1 Introduction 121
7.2 Generic considerations for the use of fluorescent indicators 122
7.3 Optimization of fluorescent light detection and background light correction 127
7.4 3-D spatial maps of fluorescent signals 129
Acknowledgements 133
References 133
Chapter Eight. Fluorescent Probes in Practice – Potential Artifacts 135
8.1 Introduction 135
8.2 Photobleaching 135
8.3 Dynamic range 136
8.4 Probe loading 137
8.5 Ion calibration 138
8.6 Cell movement and fast ion fluxes 138
8.7 Autofluorescence 139
8.8 Interactions between multiple probes 139
8.9 Averaging and intensifier noise 140
8.10 Probe leakage and exocytosis 140
8.11 Probe kinetics 140
References 140
Part IV. Optical Probes for Specific Molecules, Organelles and Cells 142
Chapter Nine. Acridine Orange as a Probe for Cell and Molecular Biology 144
9.1 Introduction 144
9.2 Historical remarks 144
9.3 AO as a fluorescent dye 145
9.4 Spectral properties of AO in complexes with nucleic acids and other biopolymers 145
9.5 AO in the study of nucleic acids in vitro 146
9.6 AO in nucleic acid cytochemistry 147
9.7 AO DNA staining after acid pretreatments 149
9.8 AO in the study of DNA thermal denaturation 149
9.9 AO in the study of the chromatin functional state 150
9.10 Fluorescence polarization of AO in studies of biopolymers 153
9.11 AO in chromosome banding 154
9.12 AO in acid polysaccharide histochemistry 154
9.13 AO binding to proteins 155
9.14 AO binding to a living cell 155
9.15 AO in the study of cell viability 158
9.16 AO in the study of apotopsis 158
9.17 AO in flow cytometry 159
9.18 Other applications of AO 159
References 159
Chapter Ten. Fluorescent Lipid Analogues: Applications in Cell and Membrane Biology 163
10.1 Introduction 163
10.2 Fluorescent lipid analogues 165
10.3 Applications 170
Acknowledgements 181
References 181
Chapter Eleven. Optical Probes for Cyclic AMP 183
11.1 Rationale for creating optical probes for cyclic AMP 183
11.2 Previous methods for measuring cAMP or imaging related molecules 184
11.3 Alternative cAMP binding sites 185
11.4 Properties of A-kinase 185
11.5 Fluorescent labelling of A-kinase 187
11.6 Properties of FlCRhR 190
11.7 Introduction of FlCRhR into cells 193
11.8 Imaging of FlCRhR and free cAMP 194
11.9 Applications 197
References 198
Part V: Technology for Qualitative and Quantitative Detection of Optical Probes in Living Cells 200
Chapter Twelve. Quantitative Digital Imaging of Biological Activity in Living Cells with Ion-sensitive Fluorescent Probes 202
12.1 Introduction 202
12.2 Fluorescent probes for living cell function 203
12.3 Observing biological activity in 'real time' 205
12.4 Ratiometric imaging of ion-sensitive flourescent probes 205
12.5 Imaging strategies 205
12.6 Digital image processing 208
12.7 Data presentation 209
12.8 Confocal laser scanning microscopy – optical approaches to enhanced image resolution 209
12.9 A novel high-speed digital confocal microscope 210
12.10 Digital deconvolution and digital confocal microscopy – 'soft' approaches to enhanced image resolution 212
12.11 Fluorescent measurements of cytosolic ions – combined photometry with electrophysiology 213
12.12 Photometric measurements in single cells 213
12.13 Measurement of fluorescent light – photometry versus imaging 214
12.14 Photomultiplier tube technology 216
12.15 Photon counting versus photocurrent integration 216
12.16 Excitation filter switching 217
12.17 Dual-emission probes 217
12.18 Electrophysiology combined with photometry or imaging 218
12.19 Data acquisition 218
12.20 CONCORD – integrating imaging, photometry and electrophysiology on a single workstation for ion imaging experiments 218
12.21 Cell culture and loading of fluorescent probes 220
12.22 Calibration of ion-sensitive dyes in living cells and in solution 220
12.23 Deriving spectra data from optical probes in situ – the SpectralWIZARD 221
12.24 Summary 222
References 222
Chapter Thirteen. Fast Photometric Measurements of Cell Function Combined with Electrophysiology 223
13.1 Fluorescent light measurement 224
13.2 A fluorescence/electrophysiological recording system 224
13.3 The photomultiplier tube 226
13.4 Dual-emission dye measurement systems 227
13.5 Dual-excitation dye measurement systems 228
13.6 Analogue signal digitization 230
13.7 Fluorescence measurement systems 230
13.8 Software for recording fluorescence signals 232
13.9 The 'chart recorder' paradigm 232
13.10 The 'oscilloscope' paradigm 232
13.11 Leak current subtraction 233
13.12 Computer system designs 234
13.13 Conclusion 235
References 235
Equipment suppliers 235
Chapter Fourteen. Potentiometric Membrane Dyes and Imaging Membrane Potential in Single Cells 237
14.1 Introduction 237
14.2 Optimization of dye indicator sensitivity 238
14.3 Mapping membrane potential by digital fluorescence microscopy 242
14.4 Conclusion 246
Acknowledgements 246
References 246
Chapter Fifteen. Fast Multisite Optical Measurement of Membrane Potential, with Two Examples 249
15.1 Introduction 249
15.2 Signal type 250
15.3 Dyes 251
15.4 Measuring technology 252
15.5 Two examples 256
15.6 Population signals from vertebrate brain 260
15.7 Future directions 262
Acknowledgements 263
References 263
Chapter Sixteen. Imaging Membrane Potential Changes in Individual Neurons 265
16.1 Introduction 265
16.2 Extracellular application of voltage-sensitive dyes 267
16.3 Intracellular application of voltage-sensitive dyes 267
16.4 Dye injection 268
16.5 Optical recording 268
16.6 Multisite recording 269
16.7 Comparison of optical and electrical signals 270
16.8 Dye sensitivity (dF/F) and signal-to-noise ratio 271
16.9 Calibration of the voltage-sensitive dye measurements in terms of membrane potential 271
16.10 Pharmacological effects and photodynamic damage 272
16.11 Imaging spike trigger zone 273
16.12 Vertebrate neurons 273
16.13 Summary 274
References 274
Part VI: Using Novel Indications for Genetic, Molecular and Cellular Function 276
Chapter Seventeen. Bioluminescent and Chemiluminescent Indicators for Molecular Signalling and Function in Living Cells 278
17.1 The natural history of bio- and chemiluminescence 278
17.2 The analytical potential of chemiluminescent compounds 280
17.3 Application of chemi- and bioluminescence to living cells 283
17.4 Bioluminescent reporter genes 288
17.5 Bioluminescent indicators for molecular signalling in live cells 293
17.6 Conclusions and future prospects 296
References 296
Chapter Eighteen. Luminescence Imaging of Gene Expression in Single Living Cells 300
18.1 Introduction 300
18.2 General considerations 301
18.3 Equipment required 303
18.4 Experimental procedures 306
18.5 Concluding remarks 309
Acknowledgements 309
References 309
Chapter Nineteen. Enhanced Variants of the Green Fluorescent Protein for Greater Sensitivity, Different Colours and Detection of Apoptosis 311
19.1 Introduction 311
19.2 GFP variants 313
19.3 Detection of apoptosis with GFP 316
References 318
Chapter Twenty. Rapid Detection of Microorganisms 320
20.1 Materials and methods 323
20.2 Results 324
20.3 Discussion 325
References 327
Part VII: Applications of Confocal Microscopy for Optical Probe Imaging 328
Chapter Twenty-One. Confocal Microscopy – Principles, Practice and Options 330
21.1 Introduction 330
21.2 Advantages of scanning 330
21.3 Confocal microscopy 331
21.4 Practical aspects 333
21.5 System performance 335
References 336
Chapter Twenty-Two. Dual-excitation Confocal Fluorescence Microscopy 337
22.1 Introduction 337
22.2 Measurement of pHi with BCECF 337
22.3 Design of a dual-excitation laser scanning confocal fluorescence microscope for measuring pHi with BCECF 339
22.4 Measurement of pHi in the cortical collecting tubule 340
22.5 Measurement of intracellular Ca 2+ in the perfused afferent arteriole 341
22.6 Future development 342
Acknowledgements 342
References 342
Chapter Twenty-Three. High-speed Confocal Imaging in Four Dimensions 343
23.1 Introduction 343
23.2 Key questions relating to data sampling 344
23.3 Noran Odyssey XL real-time confocal system 344
23.4 Spatial and temporal resolutions in 2-D imaging 345
23.5 Case studies on 2-D resolutions 348
23.6 Spatial and temporal resolutions in 3-D imaging 351
23.7 4-D temporal resolution 352
23.8 3-D and 4-D image processing, display and storage 353
23.9 Case studies on 3-D and 4-D resolutions 354
23.10 Future directions 356
Acknowledgements 357
References 357
Chapter Twenty-Four. Multiphoton Fluorescence Microscopy 358
24.1 Introduction 358
24.2 Principles of two-photon fluorescence 358
24.3 The costs and the benefits 360
24.4 Hardware for two-photon microscopy 361
Acknowledgements 362
References 362
Chapter Twenty-Five. High-resolution Confocal Imaging of Elementary Ca 2+ Signals in Living Cells 364
25.1 Calcium as an intracellular messenger 364
25.2 Investigating elementary Ca 2+ signals 366
25.3 The type of confocal microscope and the mode of scanning 368
25.4 Additional considerations 369
25.5 Summary 370
References 370
Chapter Twenty-Six. Confocal Fluorescent Microscopy Using a Nipkow Scanner 371
26.1 Introduction 371
26.2 Construction of the CSU10 372
26.3 Characteristics of the CSU10 372
26.4 System integration of the CSU10 374
26.5 Applications 374
26.6 Future prospects 375
26.7 Conclusion 376
References 376
Chapter Twenty-Seven. Optimizing Confocal Microscopy for Thick Biological Specimens 377
27.1 Introduction 377
27.2 Advantages of confocal over wide-field microscopy, especially for thick biological specimens 378
27.3 Pathlength errors 379
27.4 Dye concentration and photobleaching artifacts 380
27.5 Ratiometric analysis 380
27.6 Aberrations and confocal microscopy 381
27.7 Spherical aberration 381
27.8 Chromatic aberration 382
27.9 Avoiding refractive index mismatch 383
Acknowledgements 387
References 387
Chapter Twenty-Eight. Redox Confocal Imaging: Intrinsic Fluorescent Probes of Cellular Metabolism 388
28.1 Introduction 388
28.2 History of the use of intrinsic probes to monitor cellular metabolism 390
28.3 The biochemical basis of intrinsic fluorescent probes in living cells 391
28.4 Instrumentation for the use of low-light-level fluorescent imaging of living cells and tissues 392
28.5 Applications of intrinsic fluorescent redox probes to cellular metabolism 397
28.6 Comparison with other non-invasive techniques 399
28.7 Summary and conclusions 400
Acknowledgements 400
References 400
Part VIII: Advanced Imaging and Light Detection Approaches for Optical Probe Applications 402
Chapter Twenty-Nine. Confocal Raman Microspectrometry 404
29.1 Introduction 404
29.2 Raman spectroscopy 405
29.3 The confocal Raman microspectrometer (CRM) 409
29.4 Applications 412
29.5 Future developments 429
Acknowledgements 431
References 432
Chapter Thirty. Spectral Imaging of Autofluorescence Molecules and DNA Probes 434
30.1 Introduction 434
30.2 The SpectraCube TM system and design 435
30.3 The analysis of a spectral image 436
30.4 Applications of spectral imaging in biology 437
References 439
Chapter Thirty-One. Multiphoton Excitation Microscopy and Spectroscopy of Cells, Tissues and Human Skin In Vivo 441
31.1 Introduction 441
31.2 History of two-photon excitation microscopy 442
31.3 Physics of multiphoton excitation processes 443
31.4 Comparison with confocal microscopy 445
31.5 Instrumentation for multiphoton excitation microscopy, spectroscopy and lifetime measurement 447
31.6 Applications to cells and tissues 450
31.7 Application to in vivo functional imaging of human skin: an example of a thick, highly scattering in vivo tissue 452
31.8 Mitigation of photodamage with multiphoton excitation microscopy 455
31.9 Discussion 456
31.10 Summary and conclusions 457
Acknowledgements 457
References 457
Chapter Thirty-Two. Raman Spectroscopic Methods for In Vitro and In Vivo Tissue Characterization 460
32.1 Introduction 460
32.2 Instrumentation 461
32.3 Calibration of Raman spectra 464
32.4 Data analysis 467
32.5 Examples of tissue characterization by Raman spectroscopy 470
32.6 Conclusion 480
Acknowledgements 480
References 481
Chapter Thirty-Three. In Vivo Semiquantitative NADH-fluorescence Imaging 483
33.1 Introduction 483
33.2 Origin of UV-excited (365 nm) tissular fluorescence 484
33.3 Methodology of semiquantitative in vivo NADH-fluorescence imaging 485
33.4 Instrumentation 487
33.5 Experimental verification of the linear relationship between [NADH] and FNADH/Rdiff, 365 488
33.6 Biomedical and clinical applications of NADH-fluorescence imaging 488
33.7 Outlook 492
Acknowledgements 492
References 492
Chapter Thirty-Four. Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and Applications 494
34.1 Introduction 494
34.2 Experimental realizations of lifetime-resolved imaging microscopy 495
34.3 Implementation of a wide-field frequency-domain FLIM system 500
34.4 Example of FLIM measurement: GFP in living cells 502
34.5 Applications of FLIM 502
34.6 Concluding remarks 504
Acknowledgements 505
References 505
Chapter Thirty-Five. Detection of Flnorescently Labelled Proteins following Gel Electrophoresis: A Key Enabling Technology for Proteomics 507
35.1 Introduction 507
35.2 General application areas for fluorescent imaging in electrophoresis 507
35.3 What is electrophoresis? 508
35.4 The use of slab gels 509
35.5 Visualization of proteins 509
35.6 Fluorescent prelabelling of proteins 509
35.7 Imaging fluorescent proteins in gels 511
35.8 Alternative staining and visualization techniques 512
35.9 Imaging area 512
35.10 High-throughput acquisition 513
35.11 Examples of specific application areas 513
35.12 Summary 514
References 515
Part IX: CCD Cameras: Key Enabling Technologies for Optical Probe Imaging 516
Chapter Thirty-Six. Properties of Low-light-level Intensified Cameras 518
36.1 Introduction 518
36.2 Image intensifier: first and second generation 519
36.3 Maximizing signal and minimizing noise 520
36.4 Thermal noise 520
36.5 Non-thermal noise 520
36.6 Temporal characteristics of photocathode noise 521
36.7 Gain use and abuse 521
36.8 Spectral response 522
36.9 Comparison between second- and third-generation intensifier tubes 523
36.10 High-resolution intensifiers 524
36.11 Fibreoptics or lenses for image transfer? 524
36.12 The charge-coupled device 525
36.13 Frame and line transfer CCDs 526
36.14 Full-frame CCDs 526
36.15 CCD pixel size and dynamic range 527
36.16 Read-out features 527
36.17 High-resolution digital ICCDs 527
36.18 Fast read-out ICCDs 528
36.19 Digital ICCDs with increasing bit range 529
36.20 Analogue video ICCDs 529
36.21 Photon counting imaging 530
36.22 Systems integration 531
36.23 Flat-field normalization- the magic touch 533
Chapter Thirty-Seven. Properties of Low-light-level Slow-scan Detectors 534
37.1 Introduction 534
37.2 Contemporary image-acquisition technology 534
37.3 How CCDs work 535
37.4 The high-performance slow-scan CCD camera 537
37.5 Slow-scan CCD camera performance 538
37.6 Applications of slow-scan CCD cameras 541
37.7 Summary 543
References 543
Chapter Thirty-Eight. High-speed Digital CCD Cameras- Principles and Applications 544
38.1 Introduction 544
38.2 The need for high-speed digital CCD cameras 544
38.3 Other technological advances 546
38.4 Systems selection 549
38.5 Conclusions 551
Part X: Flow Cytometric Methodologies for Measurement of Optical Probes in Live Cells 552
Chapter Thirty-Nine. Flow Cytometry: Use of Multiparameter Kinetics to Evaluate Several Activation Parameters Simultaneously in Individual Living Cells 554
39.1 Introduction 554
39.2 Fluorescent techniques – general advantages and disadvantages 555
39.3 Flow cytometry for kinetic studies of cellular functions 556
39.4 Time of onset of initial response 556
39.5 Parameters which can be measured 557
39.6 Classes of fluorescent probes 557
39.7 Specific probes for parameters of cell function 559
39.8 Non-kinetic applications of relevance to multiparameter flow kinetics correlations 564
39.9 Conclusion 565
Acknowledgements 565
References 565
Chapter Forty. Photolabile Caged Compounds 567
40.1 Introduction 567
40.2 Properties of a photolabile probe 568
40.3 The chemistry of photolabile compounds 569
40.4 Application of photolabile probes to studying biological pathways 573
40.5 Potential problems associated with the use of caged compounds 575
40.6 Conclusion 579
Acknowledgements 579
References 579
Chapter Forty-One. Fluorescent Analogues: Optical Biosensors of the Chemical and Molecular Dynamics of Macromolecules in Living Cells 581
41.1 Introduction 581
41.2 MeroCaM 1 and 2: fluorescent indicators of calcium-calmodulin binding 582
41.3 Fluorescent analogue of myosin II 584
41.4 Protein-based optical biosensors of myosin II regulatory light chain phosphorylation 589
41.5 Future studies 591
References 592
Part XI: Applications of Optical Probe Imaging to Biological Problems 594
Chapter Forty-Two. Fluorescence and Luminescence Techniques to Probe Ion Activities in Living Plant Cells 596
42.1 Introduction 596
42.2 Tissue preparation, mounting and perfusion 597
42.3 Securing the specimen for microscopy 598
42.4 Selection and use of fluorescent probes 598
42.5 Observation and measurement of dye fluorescence 605
42.6 Calibration in vitro and in situ 608
42.7 Additional measurement techniques 611
42.8 Using recombinant aequorin for measurement of intracellular calcium in plants 612
42.9 Manipulation of intracellular events using caged probes 618
42.10 Probes for other compartments 619
42.11 Future developments 620
Acknowledgements 621
References 621
Chapter Forty-Three. Nuclear Calcium: Concepts and Controversies 624
43.1 Introduction 624
43.2 The nuclear envelope and nuclear transporters 625
43.3 Nuclear calcium 626
43.4 Summary 627
References 627
Chapter Forty-Four. Assessment of Gap Junctional Intercellular Communication in Living Cells Using Fluorescence Techniques 629
44.1 Introduction 629
44.2 Materials and methods 631
44.3 Results 633
44.4 Discussion 636
Acknowledgements 638
References 638
Chapter Forty-Five. Photoactivation of Fluorescence as a Probe for Cytoskeletal Dynamics in Mitosis and Cell Motility 640
45.1 Introduction 640
45.2 Photoactivatable fluorescent probes 642
45.3 A computer-controlled, multiple-channel fluorescence microscope for photoactivation 644
45.4 Experiments using photoactivation of fluorescence 648
45.5 Future prospects and conclusions 653
References 653
Note added in proof 654
Chapter Forty-Six. Video Imaging of Lipid Order 655
46.1 Introduction 655
46.2 Theory 656
46.3 Experiment 658
46.4 Biological applications 659
Acknowledgements 660
References 660
Index 661
Color Plate Section 676