1 Introduction to Fluorescence
1.1 Phenomena of Fluorescence ............................... 1
1.2 Jablonski Diagram ....................................... 3
1.3 Characteristics of Fluorescence Emission ................ 6
1.4 Fluorescence Lifetimes and Quantum Yields ............... 9
1.5 Fluorescence Anisotropy ................................ 12
1.6 Resonance Energy Transfer .............................. 13
1.7 Steady-State and Time-Resolved Fluorescence ............ 14
1.8 Biochemical Fluorophores ............................... 15
1.9 Molecular Information from Fluorescence ................ 17
1.10 Biochemical Examples of Basic Phenomena ................ 20
1.11 New Fluorescence Technologies .......................... 21
1.12 Overview of Fluorescence Spectroscopy .................. 24
References .................................................. 25
Problems .................................................... 25
2 Instrumentation for Fluorescence Spectroscopy
2.1. Spectrofluorometers .................................... 27
2.2 Light Sources .......................................... 31
2.3 Monochromators ......................................... 34
2.4 Optical Filters ........................................ 38
2.5 Optical Filters and Signal Purity ...................... 41
2.6 Photomultiplier Tubes .................................. 44
2.7 Polarizers ............................................. 49
2.8 Corrected Excitation Spectra ........................... 51
2.9 Corrected Emission Spectra ............................. 52
2.10 Quantum Yield Standards ................................ 54
2.11 Effects of Sample Geometry ............................. 55
2.12 Common Errors in Sample Preparation .................... 57
2.13 Absorption of Light and Deviation from the
Beer-Lambert Law ....................................... 58
2.14 Conclusions ............................................ 59
References .................................................. 59
Problems .................................................... 60
3 Fluorophores
3.1 Intrinsic or Natural Fluorophores ...................... 63
3.3 Red and Near-Infrared (NIR) Dyes ....................... 74
3.4 DNA Probes ............................................. 75
3.5 Chemical Sensing Probes ................................ 78
3.6 Special Probes ......................................... 79
3.7 Green Fluorescent Proteins ............................. 81
3.8 Other Fluorescent Proteins ............................. 83
3.9 Long-Lifetime Probes ................................... 86
3.10 Proteins as Sensors .................................... 88
3.11 Conclusion ............................................. 89
References .................................................. 90
Problems .................................................... 94
4 Time-Domain Lifetime Measurements
4.1. Overview of Time-Domain and Frequency-Domain
Measurements ........................................... 98
4.2 Biopolymers Display Multi-Exponential or
Heterogeneous Decays .................................. 101
4.3 Time-Correlated Single-Photon Counting ................ 103
4.4 Light Sources for TCSPC ............................... 107
4.5 Electronics for TCSPC ................................. 114
4.6 Detectors for TCSPC ................................... 117
4.7 Multi-Detector and Multidimensional TCSPC ............. 121
4.8 Alternative Methods for Time-Resolved Measurements .... 124
4.9 Data Analysis: Nonlinear Least Squares ................ 129
4.10 Analysis of Multi-Exponential Decays .................. 133
4.11 Intensity Decay Laws .................................. 141
4.12 Global Analysis ....................................... 144
4.13 Applications of TCSPC ................................. 145
4.14 Data Analysis: Maximum Entropy Method ................. 148
References ................................................. 149
Problems ................................................... 154
5 Frequency-Domain Lifetime Measurements
5.1 Theory of Frequency-Domain Fluorometry ................ 158
5.2 Frequency-Domain Instrumentation ...................... 163
5.3 Color Effects and Background Fluorescence ............. 168
5.4 Representative Frequency-Domain Intensity Decays ...... 170
5.5 Simple Frequency-Domain Instruments ................... 173
5.6 Gigahertz Frequency-Domain Fluorometry ................ 175
5.7 Analysis of Frequency-Domain Data ..................... 178
5.8 Biochemical Examples of Frequency-Domain Intensity
Decays ................................................ 186
5.9 Phase-Angle and Modulation Spectra ..................... 189
5.10 Apparent Phase and Modul ation Lifetimes .............. 191
5.11 Derivation of the Equations for Phase-Modulation
Fluorescence .......................................... 192
5.12 Phase-Sensitive Emission Spectra ...................... 194
5.13 Phase-Modulation Resolution of Emission Spectra ....... 197
References ................................................. 199
Problems ................................................... 203
6 Solvent and Environmental Effects
6.1 Overview of Solvent Polarity Effects .................. 205
6.2 General Solvent Effects: The Lippert-Mataga
Equation .............................................. 208
6.3 Specific Solvent Effects .............................. 213
6.4 Temperature Effects ................................... 216
6.5 Phase Transitions in Membranes ........................ 217
6.6 Additional Factors that Affect Emission Spectra ....... 219
6.7 Effects of Viscosity .................................. 223
6.8 Probe-Probe Interactions .............................. 225
6.9 Biochemical Applications of Environment-Sensitive
Fluorophores .......................................... 226
6.10 Advanced Solvent-Sensitive Probes ..................... 228
6.11 Effects of Solvent Mixtures ........................... 229
6.12 Summary of Solvent Effects ............................ 231
References ................................................. 232
Problems ................................................... 235
7 Dynamics of Solvent and Spectral Relaxation
7.1 Overview of Excited-State Processes ................... 237
7.2 Measurement of Time-Resolved Emission Spectra
(TRES) ................................................ 240
7.3 Spectral Relaxation in Proteins ....................... 242
7.4 Spectral Relaxation in Membranes ...................... 245
7.5 Picosecond Relaxation in Solvents ..................... 249
7.6 Measurement of Multi-Exponential Spectral
Relaxation ............................................ 252
7.7 Distinction between Solvent Relaxation and Formation
of Rotational Isomers ................................. 253
7.8 Comparison of TRES and Decay-Associated Spectra ....... 255
7.9 Lifetime-Resolved Emission Spectra .................... 255
7.10 Red-Edge Excitation Shifts ............................ 257
7.11 Excited-State Reactions ............................... 259
7.12 Theory for a Reversible Two-State Reaction ............ 262
7.13 Time-Domain Studies of Naphthol Dissociation .......... 264
7.14 Analysis of Excited-State Reactions by Phase-
Modulation Fluorometry ................................ 265
7.15 Biochemical Examples of Excited-State Reactions ....... 270
References ................................................. 270
Problems ................................................... 275
8 Quenching of Fluorescence
8.1 Quenchers of Fluorescence ............................. 278
8.2 Theory of Collisional Quenching ....................... 278
8.3 Theory of Static Quenching ............................ 282
8.4 Combined Dynamic and Static Quenching ................. 282
8.5 Examples of Static and Dynamic Quenching .............. 283
8.6 Deviations from the Stern-Volmer Equation: Quenching
Sphere of Action ...................................... 284
8.7 Effects of Steric Shielding and Charge on Quenching ... 286
8.8 Fractional Accessibility to Quenchers ................. 288
8.9 Applications of Quenching to Proteins ................. 290
8.10 Application of Quenching to Membranes ................. 293
8.11 Lateral Diffusion in Membranes ........................ 300
8.12 Quenching-Resolved Emission Spectra ................... 301
8.13 Quenching and Association Reactions ................... 304
8.14 Sensing Applications of Quenching ..................... 305
8.15 Applications of Quenching to Molecular Biology ........ 310
8.16 Quenching on Gold Surfaces ............................ 313
8.17 Intramolecular Quenching .............................. 314
8.18 Quenching of Phosphorescence .......................... 317
References ................................................. 318
Problems ................................................... 327
9 Mechanisms and Dynamics of Fluorescence Quenching
9.1 Comparison of Quenching and Resonance Energy
Transfer .............................................. 331
9.2 Mechanisms of Quenching ............................... 334
9.3 Energetics of Photoinduced Electron Transfer .......... 336
9.4 PET Quenching in Biomolecules ......................... 341
9.5 Single-Molecule PET ................................... 342
9.6 Transient Effects in Quenching ........................ 343
References ................................................. 348
Problems ................................................... 351
10 Fluorescence Anisotropy
10.1 Definition of Fluorescence Anisotropy ................. 353
10.2 Theory for Anisotropy ................................. 355
10.3 Excitation Anisotropy Spectra ......................... 358
10.4 Measurement of Fluorescence Anisotropics .............. 361
10.5 Effects of Rotational Diffusion on Fluorescence
Anisotropics: The Perrin Equation ..................... 366
10.6 Perrin Plots of Proteins .............................. 370
10.7 Biochemical Applications of Steady-State
Anisotropics .......................................... 372
10.8 Anisotropy of Membranes and Membrane-Bound Proteins ... 374
10.9 Transition Moments .................................... 377
References ................................................. 378
Additional Reading on the Application of Anisotropy ........ 380
Problems ................................................... 381
11 Time-Dependent Anisotropy Decays
11.1 Time-Domain and Frequency-Domain Anisotropy Decays .... 383
11.2 Anisotropy Decay Analysis ............................. 387
11.3 Analysis of Frequency-Domain Anisotropy Decays ........ 390
11.4 Anisotropy Decay Laws ................................. 390
11.5 Time-Domain Anisotropy Decays of Proteins ............. 394
11.6 Frequency-Domain Anisotropy Decays of Proteins ........ 397
11.7 Hindered Rotational Diffusion in Membranes ............ 399
11.8 Anisotropy Decays of Nucleic Acids .................... 402
11.9 Correlation Time Imaging .............................. 406
11.10 Microsecond Anisotropy Decays ........................ 408
References ................................................. 409
Problems ................................................... 412
12 Advanced Anisotropy Concepts
12.1 Associated Anisotropy Decay ........................... 413
12.2 Biochemical Examples of Associated Anisotropy
Decays ................................................ 417
12.3 Rotational Diffusion of Non-Spherical Molecules:
An Overview ........................................... 418
12.4 Ellipsoids of Revolution .............................. 420
12.5 Complete Theory for Rotational Diffusion of
Ellipsoids ............................................ 425
12.6 Anisotropic Rotational Diffusion ...................... 426
12.7 Global Anisotropy Decay Analysis ...................... 429
12.8 Intercalated Fluorophores in DNA ...................... 432
12.9 Transition Moments .................................... 433
12.10 Lifetime-Resolved Anisotropics ....................... 435
12.11 Soleillet's Rule: Multiplication of Depolarized
Factors .............................................. 436
12.12 Anisotropies Can Depend on Emission Wavelength ....... 437
References ................................................. 438
Problems ................................................... 441
13 Energy Transfer
13.1 Characteristics of Resonance Energy Transfer .......... 443
13.2 Theory of Energy Transfer for a Donor-Acceptor Pair ... 445
13.3 Distance Measurements Using RET ....................... 451
13.4 Biochemical Applications of RET ....................... 453
13.5 RET Sensors ........................................... 458
13.6 RET and Nucleic Acids ................................. 459
13.7 Energy-Transfer Efficiency from Enhanced Acceptor
Fluorescence .......................................... 461
13.8 Energy Transfer in Membranes .......................... 462
13.9 Effect of k2 on RET ................................... 465
13.10 Energy Transfer in Solution .......................... 466
13.11 Representative R0 Values ............................. 467
References ................................................. 468
Additional References on Resonance Energy Transfer ......... 471
Problems ................................................... 472
14 Time-Resolved Energy Transfer and Conformational
Distributions of Biopolymers
14.1 Distance Distributions ................................ 477
14.2 Distance Distributions in Peptides .................... 479
14.3 Distance Distributions in Peptides .................... 482
14.4 Distance-Distribution Data Analysis ................... 485
14.5 Biochemical Applications of Distance Distributions .... 490
14.6 Time-Resolved RET Imaging ............................. 497
14.7 Effect of Diffusion for Linked D-A Pairs .............. 498
14.8 Conclusion ............................................ 501
References ................................................. 501
Representative Publications on Measurement of Distance
Distributions .............................................. 504
Problems ................................................... 505
15 Energy Transfer to Multiple Acceptors in One,Two, or
Three Dimensions
15.1 RET in Three Dimensions ............................... 507
15.2 Effect of Dimensionality on RET ....................... 511
15.3 Biochemical Applications of RET with Multiple
Acceptors ............................................. 515
15.4 Energy Transfer in Restricted Geometries .............. 516
15.5 RET in the Presence of Diffusion ...................... 519
15.6 RET in the Rapid Diffusion Limit ...................... 520
15.7 Conclusions ........................................... 524
References ................................................. 524
Additional References on RET between Unlinked Donor and
Acceptor ................................................... 526
Problems ................................................... 527
16 Protein Fluorescence
16.1 Spectral Properties of the Aromatic Amino Acids ....... 530
16.2 General Features of Protein Fluorescence .............. 535
16.3 Tryptophan Emission in an Apolar Protein
Environment ........................................... 538
16.4 Energy Transfer and Intrinsic Protein Fluorescence .... 539
16.5 Calcium Binding to Calmodulin Using Phenylalanine
and Tyrosine Emission ................................. 545
16.6 Quenching of Tryptophan Residues in Proteins .......... 546
16.7 Association Reaction of Proteins ..................... 551
16.8 Spectral Properties of Genetically Engineered
Proteins .............................................. 554
16.9 Protein Folding ....................................... 557
16.10 Protein Structure and Tryptophan Emission ............ 560
16.11 Tryptophan Analogues ................................. 562
16.12 The Challenge of Protein Fluorescence ................ 566
References ................................................. 567
Problems ................................................... 573
17 Time-Resolved Protein Fluorescence
17.1 Intensity Decays of Tryptophan: The Rotamer Model ..... 578
17.2 Time-Resolved Intensity Decays of Tryptophan and
Tyrosine .............................................. 580
17.3 Intensity and Anisotropy Decays of Proteins ........... 583
17.4 Protein Unfolding Exposes the Tryptophan Residue to
Water ................................................. 588
17.5 Anisotropy Decays of Proteins ......................... 589
17.6 Biochemical Examples Using Time-Resolved Protein
Fluorescence .......................................... 591
17.7 Time-Dependent Spectral Relaxation of Tryptophan ...... 596
17.8 Phosphorescence of Proteins ........................... 598
17.9 Perspectives on Protein Fluorescence .................. 600
References ................................................. 600
Problems ................................................... 605
18 Multiphoton Excitation and Microscopy
18.1 Introduction to Multiphoton Excitation ................ 607
18.2 Cross-Sections for Multiphoton Absorption ............. 609
18.3 Two-Photon Absorption Spectra ......................... 609
18.4 Two-Photon Excitation of a DNA-Bound Fluorophore ...... 610
18.5 Anisotropies with Multiphoton Excitation .............. 612
18.6 MPE for a Membrane-Bound Fluorophore .................. 613
18.7 MPE of Intrinsic Protein Fluorescence ................. 613
18.8 Multiphoton Microscopy ................................ 616
References ................................................. 619
Problems ................................................... 621
19 Fluorescence Sensing
19.1 Optical Clinical Chemistry and Spectral Observables ... 623
19.2 Spectral Observables for Fluorescence Sensing ......... 624
19.3 Mechanisms of Sensing ................................. 626
19.4 Sensing by Collisional Quenching ...................... 627
19.5 Energy-Transfer Sensing ............................... 633
19.6 Two-State pH Sensors .................................. 637
19.7 Photoinduced Electron Transfer (PET) Probes for
Metal Ions and Anion Sensors .......................... 641
19.8 Probes of Analyte Recognition ......................... 643
19.9 Glucose-Sensitive Fluorophores ........................ 650
19.10 Protein Sensors ...................................... 651
19.11 GFP Sensors .......................................... 654
19.12 New Approaches to Sensing ............................ 655
19.13 In-Vivo Imaging ...................................... 656
19.14 Immunoassays ......................................... 658
Immunoassays ............................................... 661
References ................................................. 663
Problems ................................................... 672
20 Novel Fluorophores
20.1 Semiconductor Nanoparticles ........................... 675
20.2 Lanthanides ........................................... 679
20.3 Long-Lifetime Metal-Ligand Complexes .................. 683
20.4 Long-Wavelength Long-Lifetime Fluorophores ............ 695
References ................................................. 697
Problems ................................................... 702
21 DN A Technology
21.1 DNA Sequencing ........................................ 705
21.2 High-Sensitivity DNA Stains ........................... 712
21.3 DNA Hybridization ..................................... 715
21.4 Molecular Beacons ..................................... 720
21.5 Aptamers .............................................. 724
21.6 Multiplexed Microbead Arrays: Suspension Arrays ....... 726
21.7 Fluorescence In-Situ Hybridization .................... 727
21.8 Multicolor FISH and Spectral Karyotyping .............. 730
21.9 DNA Arrays ............................................ 732
References ................................................. 734
Problems ................................................... 740
22 Fluorescence-Lifetime Imaging Microscopy
22.1 Early Methods for Fluorescence-Lifetime Imaging ....... 743
22.2 Lifetime Imaging of Calcium Using Quin-2 .............. 744
22.3 Examples of Wide-Field Frequency-Domain FLIM .......... 746
22.4 Wide-Field FLIM Using a Gated-Image Intensifier ....... 747
22.5 Laser Scanning TCSPC FLIM ............................. 748
22.6 Frequency-Domain Laser Scanning Microscopy ............ 750
22.7 Conclusions ........................................... 752
References ................................................. 752
Additional Reading on Fluorescence-Lifetime Imaging
Microscopy ................................................. 753
Problem .................................................... 755
23 Single-Molecule Detection
23.1 Detectability of Single Molecules ..................... 759
23.2 Total Internal Reflection and Confocal Optics ......... 760
23.3 Optical Configurations for SMD ........................ 762
23.4 Instrumentation for SMD ............................... 764
23.5 Single-Molecule Photophysics .......................... 768
23.6 Biochemical Applications of SMD ....................... 770
23.7 Single-Molecule Resonance Energy Transfer ............. 773
23.8 Single-Molecule Orientation and Rotational Motions .... 775
23.9 Time-Resolved Studies of Single Molecules ............. 779
23.10 Biochemical Applications ............................. 780
23.11 Advanced Topics in SMD ............................... 784
23.12 Additional Literature on SMD ......................... 788
References ................................................. 788
Additional References on Single-Molecule Detection ......... 791
Problem .................................................... 795
24 Fluorescence Correlation Spectroscopy
24.1 Principles of Fluorescence Correlation
Spectroscopy .......................................... 798
24.2 Theory of FCS ......................................... 800
24.3 Examples of FCS Experiments ........................... 805
24.4 Applications of FCS to Bioaffinity Reactions .......... 807
24.5 FCS in Two Dimensions: Membranes ...................... 810
24.6 Effects of Intersystem Crossing ....................... 815
24.7 Effects of Chemical Reactions ......................... 816
24.8 Fluorescence Intensity Distribution Analysis .......... 817
24.9 Time-Resolved FCS ..................................... 819
24.10 Detection of Conformational Dynamics
in Macromolecules .................................... 820
24.11 FCS with Total Internal Reflection ................... 821
24.12 FCS with Two-Photon Excitation ....................... 822
24.13 Dual-Color Fluorescence Cross-Correlation
Spectroscopy ......................................... 823
24.14 Rotational Diffusion and Photo Antibunching .......... 828
24.15 Flow Measurements Using FCS .......................... 830
24.16 Additional References on FCS ......................... 832
References ................................................. 832
Additional References to FCS and Its Applications .......... 837
Problems ................................................... 840
25 Radiative Decay Engineering: Metal-Enhanced Fluorescence
25.1 Radiative Decay Engineering ........................... 841
25.2 Review of Metal Effects on Fluorescence ............... 843
25.3 Optical Properties of Metal Colloids .................. 845
25.4 Theory for Fluorophore-Colloid Interactions ........... 846
25.5 Experimental Results on Metal-Enhanced
Fluorescence .......................................... 848
25.6 Distance-Dependence of Metal-Enhanced Fluorescence .... 851
25.7 Applications of Metal-Enhanced Fluorescence ........... 851
25.8 Mechanism of MEF ...................................... 855
25.9 Perspective on RET .................................... 856
References ................................................. 856
Problem .................................................... 859
26 Radiative Decay Engineering: Surface Plasmon-Coupled
Emission
26.1 Phenomenon of SPCE .................................... 861
26.2 Surface-Plasmon Resonance ............................. 861
26.3 Expected Properties of SPCE ........................... 865
26.4 Experimental Demonstration of SPCE .................... 865
26.5 Applications of SPCE .................................. 867
26.6 Future Developments in SPCE ........................... 868
References ................................................. 870
Appendix I. Corrected Emission Spectra ...................... 873
Appendix II. Fluorescent Lifetime Standards .................. 883
Appendix III. Additional Reading .............................. 889
Index ......................................................... 923
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