30
Bernard Valeur and
Mário Nuno Berberan-Santos
Molecular Fluorescence
Further Titles of Interest
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Strehmel, B., Strehmel, V., Malpert, J. H.
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Bernard Valeur and Mário Nuno Berberan-Santos
Molecular Fluorescence
Principles and Applications
Second Edition
The Authors
Prof. Dr. Bernard ValeurConservatoire National des Arts et Métiers292 rue Saint-Martin75003 ParisFrance
Prof. Mário Nuno Berberan-SantosCentro de Química-Física MolecularInstituto Superior TécnicoAv. Rovisco Pais1049-001 LisboaPortugal
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Print ISBN: 978-3-527-32846-8ePDF ISBN: 978-3-527-65003-3oBook ISBN: 978-3-527-65000-2ePub ISBN: 978-3-527-65002-6mobi ISBN: 978-3-527-65001-9
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Contents
Preface to the First Edition XVPreface to the Second Edition XVIIAcknowledgments XIXPrologue XXI
1 Introduction 11.1 What Is Luminescence? 11.2 A Brief History of Fluorescence and Phosphorescence 2
1.2.1 Early Observations 31.2.2 On the Distinction between Fluorescence and
Phosphorescence: Decay Time Measurements 101.2.3 The Perrin–Jablonski Diagram 121.2.4 Fluorescence Polarization 141.2.5 Resonance Energy Transfer 161.2.6 Early Applications of Fluorescence 17
1.3 Photoluminescence of Organic and Inorganic Species: Fluorescence or Phosphorescence? 19
1.4 Various De-Excitation Processes of Excited Molecules 201.5 Fluorescent Probes, Indicators, Labels, and Tracers 211.6 Ultimate Temporal and Spatial Resolution: Femtoseconds,
Femtoliters, Femtomoles, and Single-Molecule Detection 23General Bibliography: Monographs and Books 25
Part I Principles 31
2 Absorption of Ultraviolet, Visible, and Near-Infrared Radiation 332.1 Electronic Transitions 332.2 Transition Probabilities: The Beer–Lambert Law, Oscillator
Strength 392.3 Selection Rules 462.4 The Franck–Condon Principle 47
V
VI Contents
2.5 Multiphoton Absorption and Harmonic Generation 49Bibliography 51
3 Characteristics of Fluorescence Emission 533.1 Radiative and Nonradiative Transitions between Electronic States 53
3.1.1 Internal Conversion 563.1.2 Fluorescence 563.1.3 Intersystem Crossing and Subsequent Processes 57
3.1.3.1 Intersystem Crossing 583.1.3.2 Phosphorescence versus Nonradiative
De-Excitation 603.1.3.3 Delayed Fluorescence 603.1.3.4 Triplet–Triplet Transitions 61
3.2 Lifetimes and Quantum Yields 613.2.1 Excited-State Lifetimes 613.2.2 Quantum Yields 643.2.3 Effect of Temperature 66
3.3 Emission and Excitation Spectra 673.3.1 Steady-State Fluorescence Intensity 673.3.2 Emission Spectra 683.3.3 Excitation Spectra 713.3.4 Stokes Shift 72
Bibliography 74
4 Structural Effects on Fluorescence Emission 754.1 Effects of the Molecular Structure of Organic Molecules on
Their Fluorescence 754.1.1 Extent of the π-Electron System: Nature of the Lowest-Lying
Transition 754.1.2 Substituted Aromatic Hydrocarbons 77
4.1.2.1 Internal Heavy Atom Effect 774.1.2.2 Electron-Donating Substituents: –OH, –OR, –NH2,
–NHR, –NR2 784.1.2.3 Electron-Withdrawing Substituents: Carbonyl and
Nitro Compounds 784.1.2.4 Sulfonates 79
4.1.3 Heterocyclic Compounds 804.1.3.1 Compounds with Heteronitrogen Atoms 804.1.3.2 Coumarins 814.1.3.3 Xanthenic Dyes 824.1.3.4 Oxazines 844.1.3.5 Cyanines 854.1.3.6 BODIPY Fluorophores 86
4.1.4 Compounds Undergoing Photoinduced ICT and Internal Rotation 87
Contents VII
4.2 Fluorescence of Conjugated Polymers (CPs) 924.3 Luminescence of Carbon Nanostructures: Fullerenes, Nanotubes, and
Carbon Dots 934.4 Luminescence of Metal Compounds, Metal Complexes, and
Metal Clusters 964.5 Luminescence of Semiconductor Nanocrystals (Quantum Dots and
Quantum Rods) 103Bibliography 105
5 Environmental Effects on Fluorescence Emission 1095.1 Homogeneous and Inhomogeneous Band Broadening – Red-Edge
Effects 1095.2 General Considerations on Solvent Effects 1105.3 Solvent Relaxation Subsequent to Photoinduced Charge
Transfer (PCT) 1125.4 Theory of Solvatochromic Shifts 1175.5 Effects of Specific Interactions 119
5.5.1 Effects of Hydrogen Bonding on Absorption and Fluorescence Spectra 119
5.5.2 Examples of Effects of Specific Interactions 1205.5.3 Polarity-Induced Inversion of n−π* and π−π*
States 1235.6 Empirical Scales of Solvent Polarity 124
5.6.1 Scales Based on Solvatochromic Shifts 1245.6.1.1 Single-Parameter Approach 1245.6.1.2 Multiparameter Approach 126
5.6.2 Scale Based on Polarity-Induced Changes in Vibronic Bands (Py Scale) 129
5.7 Viscosity Effects 1295.7.1 What is Viscosity? Significance at a Microscopic Level 1295.7.2 Viscosity Effect on the Fluorescence of Molecules Undergoing
Internal Rotations 1325.8 Fluorescence in Solid Matrices at Low Temperature 135
5.8.1 Shpol’skii Spectroscopy 1365.8.2 Matrix Isolation Spectroscopy 1375.8.3 Site-Selection Spectroscopy 137
5.9 Fluorescence in Gas Phase: Supersonic Jets 137Bibliography 138
6 Effects of Intermolecular Photophysical Processes on Fluorescence Emission 1416.1 Introduction 1416.2 Overview of the Intermolecular De-Excitation Processes of Excited
Molecules Leading to Fluorescence Quenching 1436.2.1 Phenomenological Approach 143
VIII Contents
6.2.2 Dynamic Quenching 1466.2.2.1 Stern–Volmer Kinetics 1466.2.2.2 Transient Effects 148
6.2.3 Static Quenching 1526.2.3.1 Sphere of Effective Quenching 1526.2.3.2 Formation of a Ground-State Nonfluorescent
Complex 1536.2.4 Simultaneous Dynamic and Static Quenching 1546.2.5 Quenching of Heterogeneously Emitting Systems 158
6.3 Photoinduced Electron Transfer 1596.4 Formation of Excimers and Exciplexes 162
6.4.1 Excimers 1636.4.2 Exciplexes 167
6.5 Photoinduced Proton Transfer 1686.5.1 General Equations for Deprotonation in the Excited State 1706.5.2 Determination of the Excited-State pK* 172
6.5.2.1 Prediction by Means of the Förster Cycle 1726.5.2.2 Steady-State Measurements 1736.5.2.3 Time-Resolved Experiments 174
6.5.3 pH Dependence of Absorption and Emission Spectra 1746.5.4 Equations for Bases Undergoing Protonation in the Excited
State 178Bibliography 179
7 Fluorescence Polarization: Emission Anisotropy 1817.1 Polarized Light and Photoselection of Absorbing Molecules 1817.2 Characterization of the Polarization State of Fluorescence (Polarization
Ratio and Emission Anisotropy) 1847.2.1 Excitation by Polarized Light 184
7.2.1.1 Vertically Polarized Excitation 1847.2.1.2 Horizontally Polarized Excitation 186
7.2.2 Excitation by Natural Light 1877.3 Instantaneous and Steady-State Anisotropy 187
7.3.1 Instantaneous Anisotropy 1877.3.2 Steady-State Anisotropy 188
7.4 Additivity Law of Anisotropy 1887.5 Relation between Emission Anisotropy and Angular Distribution of
the Emission Transition Moments 1907.6 Case of Motionless Molecules with Random Orientation 191
7.6.1 Parallel Absorption and Emission Transition Moments 1917.6.2 Nonparallel Absorption and Emission Transition
Moments 1927.6.3 Multiphoton Excitation 196
7.7 Effect of Rotational Motion 1997.7.1 Free Rotations 200
7.7.1.1 General Equations 200
Contents IX
7.7.1.2 Isotropic Rotations 2017.7.1.3 Anisotropic Rotations 203
7.7.2 Hindered Rotations 2067.8 Applications 207Bibliography 210
8 Excitation Energy Transfer 2138.1 Introduction 2138.2 Distinction between Radiative and Nonradiative Transfer 2188.3 Radiative Energy Transfer 2198.4 Nonradiative Energy Transfer 221
8.4.1 Interactions Involved in Nonradiative Energy Transfer 2218.4.2 The Three Main Classes of Coupling 2248.4.3 Förster’s Formulation of Long-Range Dipole–Dipole Transfer
(Very Weak Coupling) 2268.4.4 Dexter’s Formulation of Exchange Energy Transfer (Very Weak
Coupling) 2338.4.5 Selection Rules 233
8.5 Determination of Distances at a Supramolecular Level Using FRET 2358.5.1 Single Distance between the Donor and the
Acceptor 2358.5.2 Distributions of Distances in Donor–Acceptor
Pairs 2398.5.3 Single Molecule Studies 2428.5.4 On the Validity of Förster’s Theory for the Estimation of
Distances 2428.6 FRET in Ensembles of Donors and Acceptors 243
8.6.1 FRET in Three Dimensions: Effect of Viscosity 2438.6.2 Effects of Dimensionality on FRET 2478.6.3 Effects of Restricted Geometries on FRET 250
8.7 FRET between Like Molecules: Excitation Energy Migration in Assemblies of Chromophores 2508.7.1 FRET within a Pair of Like Chromophores 2518.7.2 FRET in Assemblies of Like Chromophores 2518.7.3 Lack of Energy Transfer upon Excitation at the Red Edge of the
Absorption Spectrum (Weber’s Red-Edge Effect) 2528.8 Overview of Qualitative and Quantitative Applications of FRET 252Bibliography 258
Part II Techniques 263
9 Steady-State Spectrofluorometry 2659.1 Operating Principles of a Spectrofluorometer 2659.2 Correction of Excitation Spectra 268
X Contents
9.3 Correction of Emission Spectra 2689.4 Measurement of Fluorescence Quantum Yields 2699.5 Possible Artifacts in Spectrofluorometry 271
9.5.1 Inner Filter Effects 2719.5.1.1 Excitation Inner Filter Effect 2719.5.1.2 Emission Inner Filter Effect (Self-Absorption) 2729.5.1.3 Inner Filter Effects due to the Presence of Other
Substances 2749.5.2 Autofluorescence 2749.5.3 Polarization Effects 2759.5.4 Effect of Oxygen 2759.5.5 Photobleaching Effect 276
9.6 Measurement of Steady-State Emission Anisotropy: Polarization Spectra 2779.6.1 Principles of Measurement 2779.6.2 Possible Artifacts 2799.6.3 Tests Prior to Fluorescence Polarization Measurements 279
Appendix 9.A Elimination of Polarization Effects in the Measurement of Fluorescence Intensity 281
Bibliography 283
10 Time-Resolved Fluorescence Techniques 28510.1 Basic Equations of Pulse and Phase-Modulation Fluorimetries 286
10.1.1 Pulse Fluorimetry 28610.1.2 Phase-Modulation Fluorimetry 28610.1.3 Relationship between Harmonic Response and δ-Pulse
Response 28710.1.4 General Relations for Single Exponential and
MultiExponential Decays 29010.2 Pulse Fluorimetry 292
10.2.1 Light Sources 29210.2.2 Single-Photon Timing Technique (10 ps–500 µs) 29210.2.3 Streak Camera (1 ps–10 ns) 29410.2.4 Fluorescence Upconversion (0.1–500 ps) 29510.2.5 Optical Kerr-Gating (0.1–500 ps) 297
10.3 Phase-Modulation Fluorimetry 29810.3.1 Introduction 29810.3.2 Phase Fluorimeters Using a Continuous Light Source and an
Electro-Optic Modulator 30010.3.3 Phase Fluorimeters Using the Harmonic Content of a Pulsed
Laser 30210.4 Artifacts in Time-Resolved Fluorimetry 302
10.4.1 Inner Filter Effects 30210.4.2 Dependence of the Instrument Response on
Wavelength – Color Effect 304
Contents XI
10.4.3 Polarization Effects 30410.4.4 Effects of Light Scattering 304
10.5 Data Analysis 30510.5.1 Pulse Fluorimetry 30510.5.2 Phase-Modulation Fluorimetry 30610.5.3 Judging the Quality of the Fit 30610.5.4 Global Analysis 30710.5.5 Fluorescence Decays with Underlying Distributions of Decay
Times 30810.6 Lifetime Standards 31210.7 Time-Resolved Polarization Measurements 314
10.7.1 General Equations for Time-Dependent Anisotropy and Polarized Components 314
10.7.2 Pulse Fluorimetry 31510.7.3 Phase-Modulation Fluorimetry 31710.7.4 Reference Compounds for Time-Resolved Fluorescence
Anisotropy Measurements 31810.8 Time-Resolved Fluorescence Spectra 31810.9 Lifetime-Based Decomposition of Spectra 31810.10 Comparison between Single-Photon Timing Fluorimetry and
Phase-Modulation Fluorimetry 322Bibliography 323
11 Fluorescence Microscopy 32711.1 Wide-Field (Conventional), Confocal, and Two-Photon Fluorescence
Microscopies 32811.1.1 Wide-Field (Conventional) Fluorescence
Microscopy 32811.1.2 Confocal Fluorescence Microscopy 32911.1.3 Two-Photon Excitation Fluorescence Microscopy 33111.1.4 Fluorescence Polarization Measurements in Microscopy 333
11.2 Super-Resolution (Subdiffraction) Techniques 33311.2.1 Scanning Near-Field Optical Microscopy (SNOM) 33311.2.2 Far-Field Techniques 337
11.3 Fluorescence Lifetime Imaging Microscopy (FLIM) 34011.3.1 Time-Domain FLIM 34111.3.2 Frequency-Domain FLIM 342
11.4 Applications 342Bibliography 346
12 Fluorescence Correlation Spectroscopy and Single-Molecule Fluorescence Spectroscopy 34912.1 Fluorescence Correlation Spectroscopy (FCS) 349
12.1.1 Conceptual Basis and Instrumentation 35012.1.2 Determination of Translational Diffusion Coefficients 355
XII Contents
12.1.3 Chemical Kinetic Studies 35612.1.4 Determination of Rotational Diffusion Coefficients 35912.1.5 Cross-Correlation Methods 360
12.2 Single-Molecule Fluorescence Spectroscopy 36012.2.1 General Remarks 36012.2.2 Single-Molecule Detection in Flowing Solutions 36112.2.3 Single-Molecule Detection Using Fluorescence Microscopy
Techniques 36312.2.4 Single-Molecule and Single-Particle Photophysics 36712.2.5 Applications and Usefulness of Single-Molecule
Fluorescence 371Bibliography 372
Part III Applications 377
13 Evaluation of Local Physical Parameters by Means of Fluorescent Probes 37913.1 Fluorescent Probes for Polarity 379
13.1.1 Examples of Photoinduced Charge Transfer (PCT) Probes for Polarity 380
13.1.2 Pyrene and Its Derivatives 38413.2 Estimation of “Microviscosity,” Fluidity, and Molecular Mobility 384
13.2.1 Various Methods 38513.2.2 Use of Molecular Rotors 38613.2.3 Methods Based on Intermolecular Quenching or
Intermolecular Excimer Formation 38913.2.4 Methods Based on Intramolecular Excimer Formation 39013.2.5 Fluorescence Polarization Method 393
13.2.5.1 Choice of Probes 39313.2.5.2 Homogeneous Isotropic Media 39313.2.5.3 Ordered Systems 39513.2.5.4 Practical Aspects 395
13.2.6 Concluding Remarks 39713.3 Temperature 39813.4 Pressure 402Bibliography 404
14 Chemical Sensing via Fluorescence 40914.1 Introduction 40914.2 Various Approaches of Fluorescence Sensing 41014.3 Fluorescent pH Indicators 412
14.3.1 Principles 41214.3.2 The Main Fluorescent pH Indicators 417
14.3.2.1 Coumarins 417
Contents XIII
14.3.2.2 Pyranine 41714.3.2.3 Fluorescein and Its Derivatives 41914.3.2.4 SNARF and SNAFL 41914.3.2.5 pH Indicators Based on Photoinduced Electron
Transfer (PET) 42014.4 Design Principles of Fluorescent Molecular Sensors Based on Ion or
Molecule Recognition 42014.4.1 General Aspects 42014.4.2 Recognition Units and Topology 42214.4.3 Photophysical Signal Transduction 424
14.4.3.1 Photoinduced Electron Transfer (PET) 42414.4.3.2 Photoinduced Charge Transfer (PCT) 42514.4.3.3 Excimer Formation or Disappearance 42714.4.3.4 Förster Resonance Energy Transfer
(FRET) 42714.5 Fluorescent Molecular Sensors of Metal Ions 427
14.5.1 General Aspects 42714.5.2 Fluorescent PET Cation Sensors 43014.5.3 Fluorescent PCT Cation Sensors 43014.5.4 Excimer-Based Cation Sensors 43014.5.5 Cation Sensors Based on FRET 43014.5.6 Hydroxyquinoline-Based Cation Sensors 43214.5.7 Concluding Remarks on Cation Sensors 435
14.6 Fluorescent Molecular Sensors of Anions 43614.6.1 Anion Sensors Based on Collisional Quenching 43714.6.2 Anion Sensors Based on Fluorescence Changes upon Anion
Binding 43714.6.2.1 Urea and Thiourea Groups 43814.6.2.2 Pyrrole Groups 43914.6.2.3 Polyazaalkanes 44014.6.2.4 Imidazolium Groups 44314.6.2.5 Anion Binding by Metal Ion Complexes 443
14.6.3 Anion Sensors Based on the Displacement of a Competitive Fluorescent Anionic Molecule 444
14.7 Fluorescent Molecular Sensors of Neutral Molecules 44514.7.1 Cyclodextrin-Based Fluorescent Sensors 44614.7.2 Boronic Acid-Based Fluorescent Sensors 44914.7.3 Porphyrin-Based Fluorescent Sensors 452
14.8 Fluorescence Sensing of Gases 45314.8.1 Oxygen 45314.8.2 Carbon Dioxide 45614.8.3 Nitric Oxide 45614.8.4 Explosives 456
14.9 Sensing Devices 45814.10 Remote Sensing by Fluorescence LIDAR 460
XIV Contents
14.10.1 Vegetation Monitoring 46114.10.2 Marine Monitoring 46214.10.3 Historic Monuments 462
Appendix 14.A. Spectrophotometric and Spectrofluorometric pH Titrations 462
Single-Wavelength Measurements 462Dual-Wavelength Measurements 463Appendix 14.B. Determination of the Stoichiometry and Stability Constant
of Metal Complexes from Spectrophotometric or Spectrofluorometric Titrations 465
Definition of the Equilibrium Constants 465Preliminary Remarks on Titrations by Spectrophotometry and
Spectrofluorometry 467Formation of a 1 : 1 Complex (Single-Wavelength Measurements) 467Formation of a 1 : 1 Complex (Dual-Wavelength Measurements) 469Formation of Successive Complexes ML and M2L 470Cooperativity 471Determination of the Stoichiometry of a Complex by the Method of
Continuous Variations (Job’s Method) 471Bibliography 473
15 Autofluorescence and Fluorescence Labeling in Biology and Medicine 47915.1 Introduction 47915.2 Natural (Intrinsic) Chromophores and Fluorophores 480
15.2.1 Amino Acids and Derivatives 48115.2.2 Coenzymes 48815.2.3 Chlorophylls 490
15.3 Fluorescent Proteins (FPs) 49115.4 Fluorescent Small Molecules 49315.5 Quantum Dots and Other Luminescent Nanoparticles 49715.6 Conclusion 501Bibliography 502
16 Miscellaneous Applications 50716.1 Fluorescent Whitening Agents 50716.2 Fluorescent Nondestructive Testing 50816.3 Food Science 51116.4 Forensics 51316.5 Counterfeit Detection 51416.6 Fluorescence in Art 515Bibliography 518
Appendix: Characteristics of Fluorescent Organic Compounds 521Epilogue 551Index 553
Preface to the First Edition
This book is intended for students and researchers wishing to gain a deeper understanding of molecular fluorescence, with particular reference to applications in physical, chemical, material, biological, and medical sciences.
Fluorescence was first used as an analytical tool to determine concentrations of various species, either neutral or ionic. When the analyte is fluorescent, direct determination is possible; otherwise, a variety of indirect methods using derivati-zation, formation of a fluorescent complex, or fluorescence quenching have been developed. Fluorescence sensing is the method of choice for the detection of ana-lytes with a very high sensitivity, and often has an outstanding selectivity thanks to specially designed fluorescent molecular sensors. For example, clinical diagno-sis based on fluorescence has been the object of extensive development, especially with regard to the design of optodes, that is, chemical sensors and biosensors based on optical fibers coupled with fluorescent probes (e.g., for measurement of pH, pO2, pCO2, potassium, etc., in blood).
Fluorescence is also a powerful tool for investigating the structure and dynamics of matter or living systems at a molecular or supramolecular level. Polymers, solu-tions of surfactants, solid surfaces, biological membranes, proteins, nucleic acids, and living cells are well-known examples of systems in which estimates of local parameters such as polarity, fluidity, order, molecular mobility, and electrical potential are possible by means of fluorescent molecules playing the role of probes. The latter can be intrinsic or introduced on purpose. The high sensitivity of fluori-metric methods in conjunction with the specificity of the response of probes to their microenvironment contribute toward the success of this approach. Another factor is the ability of probes to provide information on dynamics of fast phenom-ena and/or the structural parameters of the system under study.
Progress in instrumentation has considerably improved the sensitivity of fluo-rescence detection. Advanced fluorescence microscopy techniques allow detection at single molecule level, which opens up new opportunities for the development of fluorescence-based methods or assays in material sciences, biotechnology, and in the pharmaceutical industry.
The aim of this book is to give readers an overview of molecular fluorescence, allowing them to understand the fundamental phenomena and the basic tech-niques, which is a prerequisite for its practical use. The parameters that may affect
XV
XVI PrefacetotheFirstEdition
the characteristics of fluorescence emission are numerous. This is a source of richness but also of complexity. The literature is teeming with examples of errone-ous interpretations, due to a lack of knowledge of the basic principles. The reader’s attention will be drawn to the many possible pitfalls.
This book is by no means intended to be exhaustive and it should rather be considered as a textbook. Consequently, the bibliography at the end of each chapter has been restricted to a few leading papers, reviews and books in which the readers will find specific references relevant to their subjects of interest.
Fluorescence is presented in this book from the point of view of a physical chemist, with emphasis on the understanding of physical and chemical concepts. Efforts have been made to make this book easily readable by researchers and students from any scientific community. For this purpose, mathematical develop-ments have been limited to what is strictly necessary for understanding the basic phenomena. Further developments can be found in accompanying boxes for aspects of major conceptual interest. The main equations are framed so that, in a first reading, the intermediate steps can be skipped. The aim of the boxes is also to show illustrations chosen from a variety of fields. Thanks to such a presentation, it is hoped that this book will favor the relationship between various scientific communities, in particular those that are relevant to physicochemical sciences and life sciences.
I am extremely grateful to Professors Elisabeth Bardez and Mario Nuno Ber-beran-Santos for their very helpful suggestions and constant encouragement. Their critical reading of most chapters of the manuscript was invaluable. The list of colleagues and friends who should be gratefully acknowledged for their advice and encouragement would be too long, and I am afraid I would forget some of them. Special thanks are due to my son, Eric Valeur, for his help in the prepara-tion of the figures and for enjoyable discussions. I also wish to thank Professor Philip Stephens for his help in the translation of French quotations.
Finally, I will never forget that my first steps in fluorescence spectroscopy were guided by Professor Lucien Monnerie; our friendly collaboration for many years was very fruitful. I also learned much from Professor Gregorio Weber during a one-year stay in his laboratory as a postdoctoral fellow; during this wonderful experience, I met outstanding scientists and friends like Dave Jameson, Bill Man-tulin, Enrico Gratton, and many others. It is a privilege for me to belong to Weber’s “family.”
Paris, May 2001 Bernard Valeur
XVII
PrefacetotheSecondEdition
The present second edition comes out 10 years after the first one. In the interval, numerous developments of fluorescence in various fields have appeared.
Fluorescence appears to be more than ever an outstanding tool for investigating not only living cells and biological tissues but also colloids, polymers, liquid crys-tals, and so forth. In life sciences, the use of fluorescent proteins (Nobel prize 2008) and semiconductors nanocrystals as tracers are two major advances that are discussed in this new edition. Fluorescence has also become extensively used as a tool for sensing chemical species in biology, medicine, pharmaceutics, environ-ment, and food science. In addition, fluorescence determination of physical parameters (pressure, temperature, viscosity) merits discussion.
The present edition is divided into three parts: principles, techniques, and appli-cations. An appendix providing the absorption and emission characteristics of the most common fluorescent compounds has been added.
No major changes have been made in the chapters relevant to the principles, as the fundamentals of fluorescence remain the same. However, the historical section of Chapter 1 has been extended, and significant additions have been made to Chapter 4 dealing with structural effects on fluorescence.
The techniques are collected in the second part. Those that were previously considered as advanced techniques in the first edition are now currently used and are thus described in line with the more conventional techniques. Special attention has been paid to the recent developments in fluorescence microscopy, fluores-cence correlation spectroscopy, and single molecule fluorescence spectroscopy.
In the third part, applications of fluorescence are presented with emphasis on fluorescence sensing of physical parameters and chemical species. A new chapter is devoted to autofluorescence and fluorescence labeling in biology and medicine. In the last chapter, which is also new, further applications are described: whitening agents, nondestructive testing, food science, forensics, counterfeit detection, and art. All these applications show the great versatility of fluorescence and its ability to reveal what is invisible to the eye thanks to its outstanding sensitivity.
Paris, November 2011 Bernard Valeur
XIX
Acknowledgments
The authors wish to thank all their colleagues who participated in fruitful discus-sions on the various aspects of fluorescence described in this book. The list is too long to be given here.
B.V. acknowledges the Conservatoire national des arts et métiers, the Ecole normale supérieure de Cachan and the Centre national de la recherche scientifique for constant support and for providing facilities. He is very grateful to Prof. Mário N. Berberan-Santos for accepting to contribute to this second edition, and for helpful discussions.
M.N.B.S. acknowledges the Instituto Superior Técnico and Fundação para a Ciência e a Tecnologia for the facilities and financial support, and is very grateful to Prof. Bernard Valeur for his invitation, and for many years of advice and fruitful collaboration.
XXI
Prologue
La lumière joue dans notre vie un rôle essentiel: elle intervient dans la plupart de nos activités. Les Grecs de l’Antiquité le savaient bien déjà, eux qui pour dire “mourir” disaient “perdre la lumière”.
[Light plays an essential role in our lives: it is an integral part of the majority of our activities. The ancient Greeks, who for “to die” said “to lose the light”, were already well aware of this.]
Louis de Broglie, 1941
1
Introduction
Licetus, 1640 (about the Bologna stone)
1.1WhatIsLuminescence?
The word luminescence, which comes from the Latin (lumen = light) was first introduced as luminescenz by the physicist and science historian Eilhardt Wiede-mann in 1888, to describe “all those phenomena of light which are not solely conditioned by the rise in temperature,” as opposed to incandescence. Lumines-cence is often considered as cold light whereas incandescence is hot light.
Luminescence is more precisely defined as follows: spontaneous emission of radia-tion from an electronically excited species or from a vibrationally excited species not in thermal equilibrium with its environment.1) The various types of lumines-cence are classified according to the mode of excitation (see Table 1.1).
Luminescent compounds can be of very different kinds:
• Organic compounds: aromatic hydrocarbons (naphthalene, anthracene, phenan-threne, pyrene, perylene, porphyrins, phtalocyanins, etc.) and derivatives, dyes (fluorescein, rhodamines, coumarins, oxazines), polyenes, diphenylpolyenes, some amino acids (tryptophan, tyrosine, phenylalanine), etc.
• Inorganic compounds: uranyl ion (UO2+), lanthanide ions (e.g., Eu3+, Tb3+), doped
glasses (e.g., with Nd, Mn, Ce, Sn, Cu, Ag), crystals (ZnS, CdS, ZnSe, CdSe, GaS, GaP, Al2O3/Cr3+ (ruby)), semiconductor nanocrystals (e.g., CdSe), metal clusters, carbon nanotubes and some fullerenes, etc.
1
Molecular Fluorescence: Principles and Applications, Second Edition. Bernard Valeur, Mário Nuno Berberan-Santos.© 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
1) Braslavsky, S. et al. (2007) Glossary of terms used in photochemistry, Pure Appl. Chem., 79, 293–465.
. . . ex arte calcinati, et illuminato aeri seu solis radiis, seu flammae fulgoribus expositi, lucem inde sine calore concipiunt in sese; . . .
[. . . properly calcinated, and illuminated either by sunlight or flames, they conceive light from themselves without heat; . . .]
2 1 Introduction
• Organometallic compounds: porphyrin metal complexes, ruthenium complexes (e.g., Ru bpy( ) +
32 ), copper complexes, complexes with lanthanide ions, com-
plexes with fluorogenic chelating agents (e.g., 8-hydroxy-quinoline, also called oxine), etc.
Fluorescence and phosphorescence are particular cases of luminescence (Table 1.1). The mode of excitation is absorption of one or more photons, which brings the absorbing species into an electronic excited state. The spontaneous emission of photons accompanying de-excitation is then called photoluminescence which is one of the possible physical effects resulting from interaction of light with matter, as shown in Figure 1.1. Stimulated emission of photons can also occur under certain conditions (see Chapter 3, Box 3.2). Additional processes, not shown, can take place for extremely high intensities of radiation, but are not relevant for luminescence studies.
1.2ABriefHistoryofFluorescenceandPhosphorescence
It is worth giving a brief account of the history of fluorescence and phosphores-cence. The major events from the early stages to the middle of the twentieth century are reported in Table 1.2 together with the names of the associated scien-tists. The story of fluorescence started with a report by N. Monardes in 1565, but scientists focused their attention on light emission phenomena other than incan-descence only in the nineteenth century. However, the major experimental and theoretical aspects of fluorescence and phosphorescence were really understood
Table1.1 Thevarioustypesofluminescence.
Phenomenon Modeofexcitation
Photoluminescence (fluorescence, phosphorescence, delayed fluorescence)
Absorption of light (photons)
Radioluminescence Ionizing radiation (X-rays, α, β, γ )
Cathodoluminescence Cathode rays (electron beams)
Electroluminescence Electric field
Thermoluminescence Heating after prior storage of energy (e.g., radioactive irradiation)
Chemiluminescence Chemical reaction (e.g., oxidation)
Bioluminescence In vivo biochemical reaction
Triboluminescence Frictional and electrostatic forces
Sonoluminescence Ultrasound
1.2 ABriefHistoryofFluorescenceandPhosphorescence 3
Figure1.1 Positionofphotoluminescenceintheframeoflight–matterinteractions.
Light-matterinteractions
• Elastic (Rayleigh) I l ti (R B ill i ) • Inelastic aman, r ouin
Ph t l i
Stimulatedemission
Spontaneousemission
AbsorptionScattering Ionization
= otoluminescence
ElectroluminescenceThermoluminescenceCh il iChemiluminescenceBioluminescenceetc.
Luminescence
only after the emergence of quantum theory, already in the twentieth century (1918–1935, i.e., less than 20 years). As in many other areas of theoretical physics and chemistry, this was an exceptionally fecund period.
1.2.1EarlyObservations
Let us examine first the origins of the terms fluorescence and phosphorescence. The term phosphorescence comes from the Greek: φως = light (genitive case: φoτoς → photon) and φoρειν = to bear (Scheme 1.1). Therefore, phosphor means “which bears light.” The term phosphor has indeed been assigned since the Middle Ages to materials that glow in the dark after exposure to light. There are many examples of minerals reported a long time ago that exhibit this property, and the most famous of them (but not the first one) was the Bolognian phosphor discov-ered by a cobbler from Bologna in 1602, Vincenzo Cascariolo, whose hobby was alchemy. One day he went for a walk in the Monte Paterno area and he picked up some strange heavy stones. After calcination with coal, he observed that these stones glowed in the dark after exposure to light. It was recognized later that the
Scheme1.1
phosphor = which bears light
Photonφοτος (Genitive case)
φορειν= to bear
Phosphorescence
φως= light
4 1 Introduction
Table1.2 Milestonesinthehistoryoffluorescenceandphosphorescencea).
Year Scientist Observationorachievement
1565 N. Monardes Emission of light by an infusion of the wood later called Lignum nephriticum (first report on the observation of fluorescence)
1602 V. Cascariolo Emission of light by Bolognese stone (first detailed observation of phosphorescence)
1640 Licetus Study of Bolognian stone. First definition as a nonthermal light emission
1833 D. Brewster Emission of light by chlorophyll solutions and fluorspar crystals
1842 J. Herschel Emission of light by quinine sulfate solutions (epipolic dispersion)
1845 E. Becquerel Emission of light by calcium sulfide upon excitation in the UV
First statement that the emitted light is of longer wavelength than the incident light.
1852 G. G. Stokes Emission of light by quinine sulfate solutions upon excitation in the UV (refrangibility of light)
1853 G. G. Stokes Introduction of the term fluorescence
1858 E. Becquerel First phosphoroscope. First lifetime measurements.
1867 F. Goppelsröder First fluorometric analysis (determination of Al(III) by the fluorescence of its morin chelate)
1871 A. Von Baeyer Synthesis of fluorescein
1888 E. Wiedemann Introduction of the term luminescence
1905, 1910 E. L. Nichols and E. Merrit First fluorescence excitation spectrum of a dye
1907 E.L. Nichols and E. Merrit Mirror symmetry between absorption and fluorescence spectra
1919 O. Stern and M. Volmer Relation for fluorescence quenching
1920 F. Weigert Discovery of the polarization of the fluorescence emitted by dye solutions
1922 S. I. Vavilov Excitation-wavelength independence of the fluorescence quantum yield
1923 S. I. Vavilov and W. L. Levshin
First study of the fluorescence polarization of dye solutions
1924 S. I. Vavilov First determination of fluorescence yield of dye solutions
1924 F. Perrin Quantitative description of static quenching (active sphere model
1924 F. Perrin First observation of alpha phosphorescence (E-type delayed fluorescence)
1925 F. Perrin Theory of fluorescence polarization (influence of viscosity)
1.2 ABriefHistoryofFluorescenceandPhosphorescence 5
Year Scientist Observationorachievement
1925 W. L. Levshin Theory of polarized fluorescence and phosphorescence
1925 J. Perrin Introduction of the term delayed fluorescence
Prediction of long-range energy transfer
1926 E. Gaviola First direct measurement of nanosecond lifetimes by phase fluorometry (instrument built in Pringsheim’s laboratory)
1926 F. Perrin Theory of fluorescence polarization (sphere)
Perrin’s equation
Indirect determination of lifetimes in solution.
Comparison with radiative lifetimes
1927 E. Gaviola and P. Pringsheim
Demonstration of resonance energy transfer in solutions
1928 E. Jette and W. West First photoelectric fluorometer
1929 F. Perrin Discussion on Jean Perrin’s diagram for the explanation of the delayed fluorescence by the intermediate passage through a metastable state
First qualitative theory of fluorescence depolarization by resonance energy transfer
1929 J. Perrin and N. Choucroun
Sensitized dye fluorescence due to energy transfer
1932 F. Perrin Quantum mechanical theory of long-range energy transfer between atoms
1934 F. Perrin Theory of fluorescence polarization (ellipsoid)
1935 A. Jablonski Jablonski’s diagram
1943 A. Terenin Triplet state
1944 G. Lewis and M. Kasha Triplet state
1946–1948 Th. Förster Theory of resonance energy transfer via dipole–dipole interaction
a) More details can be found in the following:Harvey, E.N. (1957) History of Luminescence, The American Philosophical Society, Philadelphia.O’Haver, T.C. (1978) The development of luminescence spectrometry as an analytical tool, J. Chem. Educ., 55, 423–8.Nickel, B. (1996) From the Perrin diagram to the Jablonski diagram. EPA Newslett., 58 (Part 1), 9–38.Nickel, B. (1997) From the Perrin diagram to the Jablonski diagram. EPA Newslett., 61 (Part 2), 27–60.Nickel, B. (1998) From Wiedemann’s discovery to the Jablonski diagram. EPA Newslett., 64, 19–72.Berberan-Santos, M.N. (2001) Pioneering contributions of Jean and Francis Perrin to molecular fluorescence, in New Trends in Fluorescence Spectroscopy. Applications to Chemical and Life Sciences (eds B. Valeur and J.C. Brochon), Springer-Verlag, Berlin, pp. 7–33.Valeur, B. and Berberan-Santos, M.N. (2011), A brief history of fluorescence and phosphorescence before the emergence of quantum theory, J. Chem. Educ., 88, 731–738.
Table1.2 (Continued)
6 1 Introduction
stones contained barium sulfate, which, upon reduction by coal, led to barium sulfide, a phosphorescent compound. Later, the same name phosphor was assigned to the element isolated by Brandt in 1677 (despite the fact that it is chemically very different) because, when exposed to air, it burns and emits vapors that glow in the dark.
In contrast to phosphorescence, the etymology of the term fluorescence is not at all obvious. It is indeed strange, at first sight, that this term contains fluor which is not remarked by its fluorescence! The term fluorescence was introduced by Sir George Gabriel Stokes, a physicist and professor of mathematics at Cambridge in the middle of the nineteenth century. Before explaining why Stokes coined this term, it should be recalled that the first printed observation of fluorescence was made by a Spanish physician, Nicolas Monardes, in 1565. He reported the wonder-ful peculiar blue color (under certain conditions of observation, Figure 1.2) of an infusion of a wood brought from Mexico used to treat kidney and urinary diseases: palo para los males de los riñones, y de urina (later called Lignum nephriticum).
This wood, whose peculiar color effect and diuretic properties were already known to the Aztecs, was a scarce and expensive medicine. Therefore, it was of
Figure1.2 AbsorptionandfluorescencecolorsofaninfusionofLignumnephriticumunderdaylight.(a)takenfromSafford,W.E.(1915)Ann.Rep.SmithsonianInst.,1915,
271–298.(b)mildlyalkalineaqueoussolutiontowhichchipsofEysenhardtiapolys-tachya–kindlyprovidedbyDr.A.U.Acuña–wereadded.
a)
b)
