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Molecular Fluorescence: Principles and Applications, 2nd Edition

ISBN: 978-3-527-32837-6
592 pages
May 2012
Molecular Fluorescence: Principles and Applications, 2nd Edition (3527328378) cover image
This second edition of the well-established bestseller is completely updated and revised with approximately 30 % additional material, including two new chapters on applications, which has seen the most significant developments.

The comprehensive overview written at an introductory level covers fundamental aspects, principles of instrumentation and practical applications, while providing many valuable tips.

For photochemists and photophysicists, physical chemists, molecular physicists, biophysicists, biochemists and biologists, lecturers and students of chemistry, physics, and biology.
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Preface to the First Edition XV

Preface to the Second Edition XVII

Acknowledgments XIX

Prologue XXI

1 Introduction 1

1.1 What Is Luminescence? 1

1.2 A Brief History of Fluorescence and Phosphorescence 2

1.3 Photoluminescence of Organic and Inorganic Species: Fluorescence or Phosphorescence? 19

1.4 Various De-Excitation Processes of Excited Molecules 20

1.5 Fluorescent Probes, Indicators, Labels, and Tracers 21

1.6 Ultimate Temporal and Spatial Resolution: Femtoseconds, Femtoliters, Femtomoles, and Single-Molecule Detection 23

General Bibliography: Monographs and Books 25

Part I Principles 31

2 Absorption of Ultraviolet, Visible, and Near-Infrared Radiation 33

2.1 Electronic Transitions 33

2.2 Transition Probabilities: The Beer–Lambert Law, Oscillator Strength 39

2.3 Selection Rules 46

2.4 The Franck–Condon Principle 47

2.5 Multiphoton Absorption and Harmonic Generation 49

Bibliography 51

3 Characteristics of Fluorescence Emission 53

3.1 Radiative and Nonradiative Transitions between Electronic States 53

3.2 Lifetimes and Quantum Yields 61

3.3 Emission and Excitation Spectra 67

Bibliography 74

4 Structural Effects on Fluorescence Emission 75

4.1 Effects of the Molecular Structure of Organic Molecules on Their Fluorescence 75

4.2 Fluorescence of Conjugated Polymers (CPs) 92

4.3 Luminescence of Carbon Nanostructures: Fullerenes, Nanotubes, and Carbon Dots 93

4.4 Luminescence of Metal Compounds, Metal Complexes, and Metal Clusters 96

4.5 Luminescence of Semiconductor Nanocrystals (Quantum Dots and Quantum Rods) 103

Bibliography 105

5 Environmental Effects on Fluorescence Emission 109

5.1 Homogeneous and Inhomogeneous Band Broadening – Red-Edge Effects 109

5.2 General Considerations on Solvent Effects 110

5.3 Solvent Relaxation Subsequent to Photoinduced Charge Transfer (PCT) 112

5.4 Theory of Solvatochromic Shifts 117

5.5 Effects of Specifi c Interactions 119

5.6 Empirical Scales of Solvent Polarity 124

5.7 Viscosity Effects 129

5.8 Fluorescence in Solid Matrices at Low Temperature 135

5.9 Fluorescence in Gas Phase: Supersonic Jets 137

Bibliography 138

6 Effects of Intermolecular Photophysical Processes on Fluorescence Emission 141

6.1 Introduction 141

6.2 Overview of the Intermolecular De-Excitation Processes of Excited Molecules Leading to Fluorescence Quenching 143

6.3 Photoinduced Electron Transfer 159

6.4 Formation of Excimers and Exciplexes 162

6.5 Photoinduced Proton Transfer 168

Bibliography 179

7 Fluorescence Polarization: Emission Anisotropy 181

7.1 Polarized Light and Photoselection of Absorbing Molecules 181

7.2 Characterization of the Polarization State of Fluorescence (Polarization Ratio and Emission Anisotropy) 184

7.3 Instantaneous and Steady-State Anisotropy 187

7.4 Additivity Law of Anisotropy 188

7.5 Relation between Emission Anisotropy and Angular Distribution of the Emission Transition Moments 190

7.6 Case of Motionless Molecules with Random Orientation 191

7.7 Effect of Rotational Motion 199

7.8 Applications 207

Bibliography 210

8 Excitation Energy Transfer 213

8.1 Introduction 213

8.2 Distinction between Radiative and Nonradiative Transfer 218

8.3 Radiative Energy Transfer 219

8.4 Nonradiative Energy Transfer 221

8.5 Determination of Distances at a Supramolecular Level Using FRET 235

8.6 FRET in Ensembles of Donors and Acceptors 243

8.7 FRET between Like Molecules: Excitation Energy Migration in Assemblies of Chromophores 250

8.8 Overview of Qualitative and Quantitative Applications of FRET 252

Bibliography 258

Part II Techniques 263

9 Steady-State Spectrofl uorometry 265

9.1 Operating Principles of a Spectrofl uorometer 265

9.2 Correction of Excitation Spectra 268

9.3 Correction of Emission Spectra 268

9.4 Measurement of Fluorescence Quantum Yields 269

9.5 Possible Artifacts in Spectrofluorometry 271

9.6 Measurement of Steady-State Emission Anisotropy: Polarization Spectra 277

Appendix 9.A Elimination of Polarization Effects in the Measurement of Fluorescence Intensity 281

Bibliography 283

10 Time-Resolved Fluorescence Techniques 285

10.1 Basic Equations of Pulse and Phase-Modulation Fluorimetries 286

10.2 Pulse Fluorimetry 292

10.3 Phase-Modulation Fluorimetry 298

10.4 Artifacts in Time-Resolved Fluorimetry 302

10.5 Data Analysis 305

10.6 Lifetime Standards 312

10.7 Time-Resolved Polarization Measurements 314

10.8 Time-Resolved Fluorescence Spectra 318

10.9 Lifetime-Based Decomposition of Spectra 318

10.10 Comparison between Single-Photon Timing Fluorimetry and Phase-Modulation Fluorimetry 322

Bibliography 323

11 Fluorescence Microscopy 327

11.1 Wide-Field (Conventional), Confocal, and Two-Photon Fluorescence Microscopies 328

11.2 Super-Resolution (Subdiffraction) Techniques 333

11.3 Fluorescence Lifetime Imaging Microscopy (FLIM) 340

11.4 Applications 342

Bibliography 346

12 Fluorescence Correlation Spectroscopy and Single-Molecule Fluorescence Spectroscopy 349

12.1 Fluorescence Correlation Spectroscopy (FCS) 349

12.2 Single-Molecule Fluorescence Spectroscopy 360

Bibliography 372

Part III Applications 377

13 Evaluation of Local Physical Parameters by Means of Fluorescent Probes 379

13.1 Fluorescent Probes for Polarity 379

13.2 Estimation of “Microviscosity,” Fluidity, and Molecular Mobility 384

13.3 Temperature 398

13.4 Pressure 402

Bibliography 404

14 Chemical Sensing via Fluorescence 409

14.1 Introduction 409

14.2 Various Approaches of Fluorescence Sensing 410

14.3 Fluorescent pH Indicators 412

14.4 Design Principles of Fluorescent Molecular Sensors Based on Ion or Molecule Recognition 420

14.5 Fluorescent Molecular Sensors of Metal Ions 427

14.6 Fluorescent Molecular Sensors of Anions 436

14.7 Fluorescent Molecular Sensors of Neutral Molecules 445

14.8 Fluorescence Sensing of Gases 453

14.9 Sensing Devices 458

14.10 Remote Sensing by Fluorescence LIDAR 460

Appendix 14.A. Spectrophotometric and Spectrofluorometric pH Titrations 462

Single-Wavelength Measurements 462

Dual-Wavelength Measurements 463

Appendix 14.B. Determination of the Stoichiometry and Stability Constant of Metal Complexes from Spectrophotometric or Spectrofl uorometric Titrations 465

Definition of the Equilibrium Constants 465

Preliminary Remarks on Titrations by Spectrophotometry and Spectrofl uorometry 467

Formation of a 1 : 1 Complex (Single-Wavelength Measurements) 467

Formation of a 1 : 1 Complex (Dual-Wavelength Measurements) 469

Formation of Successive Complexes ML and M2L 470

Cooperativity 471

Determination of the Stoichiometry of a Complex by the Method of Continuous Variations (Job’s Method) 471

Bibliography 473

15 Autofluorescence and Fluorescence Labeling in Biology and Medicine 479

15.1 Introduction 479

15.2 Natural (Intrinsic) Chromophores and Fluorophores 480

15.3 Fluorescent Proteins (FPs) 491

15.4 Fluorescent Small Molecules 493

15.5 Quantum Dots and Other Luminescent Nanoparticles 497

15.6 Conclusion 501

Bibliography 502

16 Miscellaneous Applications 507

16.1 Fluorescent Whitening Agents 507

16.2 Fluorescent Nondestructive Testing 508

16.3 Food Science 511

16.4 Forensics 513

16.5 Counterfeit Detection 514

16.6 Fluorescence in Art 515

Bibliography 518

Appendix: Characteristics of Fluorescent Organic Compounds 521

Epilogue 551

Index 553

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Bernard Valeur received his engineering diploma from the École Supérieure de Physique et de Chimie Industrielles de Paris (E.S.P.C.I.) and his PhD degree from the Université Pierre-et-Marie-Curie (Paris, France), followed by postdoctoral research at the University of Illinois at Urbana-Champaign (USA). After being an associate professor at E.S.P.C.I, he became full professor of physical chemistry at the Conservatoire National des Arts et Métiers (Paris) in 1979, where he is emeritus professor since 2008. Professor Valeur is a member of the laboratory Photophysique et Photochimie Supramoléculaires et Macromoléculaires at the École Normale Supérieure de Cachan since 1996. From 1995 to 2000 he served as an elected member of the French Comité National de la Recherche Scientifique. He is the author of over 170 articles or book chapters, five books, and the editor of one book. In addition, he is a member of several editorial boards.


Mário Nuno Berberan-Santos graduated in chemical engineering from Instituto Superior Técnico (IST, Technical University of Lisbon, Portugal). After a brief stay at the National Research Council of Canada (Ottawa), he received his PhD in chemistry from IST in 1989. He was a post-doctoral fellow with Bernard Valeur at Conservatoire National des Arts et Métiers (Paris, France), and at Laboratoire pour l'Utilisation du Rayonnement Electromagnétique (Univ. Paris-Sud, Orsay, France). He is full professor of Physical Chemistry at IST, and was invited full professor at the École Normale Supérieure de Cachan (France). He is a member of several editorial advisory boards and is president of the Portuguese Chemical Society (2010-2012). He has authored over 180 publications, including 150 papers in scientific journals, several book chapters, and was the editor of one book.
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This second edition of the well-established bestseller is completely updated and revised with approximately 30 % additional material.

The chapters coavering the principles and the techniques of the method (chapters 2-6) have been expanded and changed where clarification is needed.

Significant additions have been made on applications, including two completely new chapters.
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“Molecular Fluorescence is a comprehensive textbook and reference book that provides a deep and critical analysis of this evolving field.”  (Journal of Biomedical Optics, 1 March 2013)

"The strength of the book lies in its clear and understandable presentation, and in the thoroughness of the descriptions of fluorescence applications, enabling one to quickly appreciate the many questions and problems in the field of fluorescence. Molecular Fluorescence is more a textbook than a monograph, and therefore it is of special interest for students and beginners in the field, and be recommended."
- Angewandte Chemie (international edition), 2002; Vol. 41 No. 16
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