molecular fluorescence principles and applications

Bernard Valeur Molecular Fluorescence Principles and Applications Molecular Fluorescence: Principles and Applications. Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic) Related Titles from WILEY-VCH Broekaert, J. A. C. Analytical Atomic Spectrometry with Flames and Plasmas 2001. isbn 3-527-30146-1 G € uunzler, H. and Williams, A. Handbook of Analytical Techniques 2 Volumes 2001. isbn 3-527-30165-8 Feringa, B. L. Molecular Switches 2001. isbn 3-527-29965-3 Zander, C.; Keller, R. A. and Enderlein, J. Single-Molecule Detection in Solution. Methods and Applications 2002. isbn 3-527-40310-8 Molecular Fluorescence: Principles and Applications. Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic) Bernard Valeur Molecular Fluorescence Principles and Applications Weinheim – New York – Chichester – Brisbane – Singapore – Toronto Molecular Fluorescence: Principles and Applications. Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic) Prof. Dr. Bernard Valeur Laboratoire de Chimie Ge ´ ne ´ rale Conservatoire National des Arts et Me ´ tiers 292 rue Saint-Martin 75141 Paris Cedex 03 France Cover The photograph was provided by Prof. R. Clegg (University of Illinois, USA). 9 This book was carefully produced. Nevertheless, author and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek – CIP Cataloguing- in-Publication-Data A catalogue record for this publication is available from Die Deutsche Bibliothek ( WILEY-VCH Verlag GmbH, 69469 Weinheim (Federal Republic of Germany). 2002 All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany. Printed on acid-free paper. Typesetting Asco Typesetters, Hongkong Printing betz-druck gmbh, Darm-stadt Bookbinding J. Scha ¨ ffer GmbH&Co. KG, Gru ¨ nstadt ISBN 3-527-29919-X Molecular Fluorescence: Principles and Applications. Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic) Contents Preface xii Prologue 1 1 Introduction 3 1.1 What is luminescence? 3 1.2 A brief history of fluorescence and phosphorescence 5 1.3 Fluorescence and other de-excitation processes of excited molecules 8 1.4 Fluorescent probes 11 1.5 Molecular fluorescence as an analytical tool 15 1.6 Ultimate spatial and temporal resolution: femtoseconds, femtoliters, femtomoles and single-molecule detection 16 1.7 Bibliography 18 2 Absorption of UV–visible light 20 2.1 Types of electronic transitions in polyatomic molecules 20 2.2 Probability of transitions. The Beer–Lambert Law. Oscillator strength 23 2.3 Selection rules 30 2.4 The Franck–Condon principle 30 2.5 Bibliography 33 3 Characteristics of fluorescence emission 34 3.1 Radiative and non-radiative transitions between electronic states 34 3.1.1 Internal conversion 37 3.1.2 Fluorescence 37 3.1.3 Intersystem crossing and subsequent processes 38 3.1.3.1 Intersystem crossing 41 3.1.3.2 Phosphorescence versus non-radiative de-excitation 41 3.1.3.3 Delayed fluorescence 41 3.1.3.4 Triplet–triplet transitions 42 3.2 Lifetimes and quantum yields 42 3.2.1 Excited-state lifetimes 42 Molecular Fluorescence: Principles and Applications. Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic) v 3.2.2 Quantum yields 46 3.2.3 Effect of temperature 48 3.3 Emission and excitation spectra 48 3.3.1 Steady-state fluorescence intensity 48 3.3.2 Emission spectra 50 3.3.3 Excitation spectra 52 3.3.4 Stokes shift 54 3.4 Effects of molecular structure on fluorescence 54 3.4.1 Extent of p-electron system. Nature of the lowest-lying transition 54 3.4.2 Substituted aromatic hydrocarbons 56 3.4.2.1 Internal heavy atom effect 56 3.4.2.2 Electron-donating substituents: aOH, aOR, aNHR, aNH 2 56 3.4.2.3 Electron-withdrawing substituents: carbonyl and nitro compounds 57 3.4.2.4 Sulfonates 58 3.4.3 Heterocyclic compounds 59 3.4.4 Compounds undergoing photoinduced intramolecular charge transfer (ICT) and internal rotation 62 3.5 Environmental factors affecting fluorescence 67 3.5.1 Homogeneous and inhomogeneous broadening. Red-edge effects 67 3.5.2 Solid matrices at low temperature 68 3.5.3 Fluorescence in supersonic jets 70 3.6 Bibliography 70 4 Effects of intermolecular photophysical processes on fluorescence emission 72 4.1 Introduction 72 4.2 Overview of the intermolecular de-excitation processes of excited molecules leading to fluorescence quenching 74 4.2.1 Phenomenological approach 74 4.2.2 Dynamic quenching 77 4.2.2.1 Stern–Volmer kinetics 77 4.2.2.2 Transient effects 79 4.2.3 Static quenching 84 4.2.3.1 Sphere of effective quenching 84 4.2.3.2 Formation of a ground-state non-fluorescent complex 85 4.2.4 Simultaneous dynamic and static quenching 86 4.2.5 Quenching of heterogeneously emitting systems 89 4.3 Photoinduced electron transfer 90 4.4 Formation of excimers and exciplexes 94 4.4.1 Excimers 94 4.4.2 Exciplexes 99 4.5 Photoinduced proton transfer 99 4.5.1 General equations 100 4.5.2 Determination of the excited-state pK à 103 vi Contents 4.5.2.1 Prediction by means of the Fo ¨ rster cycle 103 4.5.2.2 Steady-state measurements 105 4.5.2.3 Time-resolved experiments 106 4.5.3 pH dependence of absorption and emission spectra 106 4.6 Excitation energy transfer 110 4.6.1 Distinction between radiative and non-radiative transfer 110 4.6.2 Radiative energy transfer 110 4.6.3 Non-radiative energy transfer 113 4.7 Bibliography 123 5 Fluorescence polarization. Emission anisotropy 125 5.1 Characterization of the polarization state of fluorescence (polarization ratio, emission anisotropy) 127 5.1.1 Excitation by polarized light 129 5.1.1.1 Vertically polarized excitation 129 5.1.1.2 Horizontally polarized excitation 130 5.1.2 Excitation by natural light 130 5.2 Instantaneous and steady-state anisotropy 131 5.2.1 Instantaneous anisotropy 131 5.2.2 Steady-state anisotropy 132 5.3 Additivity law of anisotropy 132 5.4 Relation between emission anisotropy and angular distribution of the emission transition moments 134 5.5 Case of motionless molecules with random orientation 135 5.5.1 Parallel absorption and emission transition moments 135 5.5.2 Non-parallel absorption and emission transition moments 138 5.6 Effect of rotational Brownian motion 140 5.6.1 Free rotations 143 5.6.2 Hindered rotations 150 5.7 Applications 151 5.8 Bibliography 154 6 Principles of steady-state and time-resolved fluorometric techniques 155 6.1 Steady-state spectrofluorometry 155 6.1.1 Operating principles of a spectrofluorometer 156 6.1.2 Correction of excitation spectra 158 6.1.3 Correction of emission spectra 159 6.1.4 Measurement of fluorescence quantum yields 159 6.1.5 Problems in steady-state fluorescence measurements: inner filter effects and polarization effects 161 6.1.6 Measurement of steady-state emission anisotropy. Polarization spectra 165 6.2 Time-resolved fluorometry 167 6.2.1 General principles of pulse and phase-modulation fluorometries 167 Contents vii 6.2.2 Design of pulse fluorometers 173 6.2.2.1 Single-photon timing technique 173 6.2.2.2 Stroboscopic technique 176 6.2.2.3 Other techniques 176 6.2.3 Design of phase-modulation fluorometers 177 6.2.3.1 Phase fluorometers using a continuous light source and an electro-optic modulator 178 6.2.3.2 Phase fluorometers using the harmonic content of a pulsed laser 180 6.2.4 Problems with data collection by pulse and phase-modulation fluorometers 180 6.2.4.1 Dependence of the instrument response on wavelength. Color effect 180 6.2.4.2 Polarization effects 181 6.2.4.3 Effect of light scattering 181 6.2.5 Data analysis 181 6.2.5.1 Pulse fluorometry 181 6.2.5.2 Phase-modulation fluorometry 182 6.2.5.3 Judging the quality of the fit 183 6.2.5.4 Global analysis 184 6.2.5.5 Complex fluorescence decays. Lifetime distributions 185 6.2.6 Lifetime standards 186 6.2.7 Time-dependent anisotropy measurements 189 6.2.7.1 Pulse fluorometry 189 6.2.7.2 Phase-modulation fluorometry 192 6.2.8 Time-resolved fluorescence spectra 192 6.2.9 Lifetime-based decomposition of spectra 194 6.2.10 Comparison between pulse and phase fluorometries 195 6.3 Appendix: Elimination of polarization effects in the measurement of fluorescence intensity and lifetime 196 6.4 Bibliography 198 7 Effect of polarity on fluorescence emission. Polarity probes 200 7.1 What is polarity? 200 7.2 Empirical scales of solvent polarity based on solvatochromic shifts 202 7.2.1 Single-parameter approach 202 7.2.2 Multi-parameter approach 204 7.3 Photoinduced charge transfer (PCT) and solvent relaxation 206 7.4 Theory of solvatochromic shifts 208 7.5 Examples of PCT fluorescent probes for polarity 213 7.6 Effects of specific interactions 217 7.6.1 Effects of hydrogen bonding on absorption and fluorescence spectra 218 7.6.2 Examples of the effects of specific interactions 218 7.6.3 Polarity-induced inversion of n–p à and p–p à states 221 7.7 Polarity-induced changes in vibronic bands. The Py scale of polarity 222 viii Contents 7.8 Conclusion 224 7.9 Bibliography 224 8 Microviscosity, fluidity, molecular mobility. Estimation by means of fluorescent probes 226 8.1 What is viscosity? Significance at a microscopic level 226 8.2 Use of molecular rotors 230 8.3 Methods based on intermolecular quenching or intermolecular excimer formation 232 8.4 Methods based on intramolecular excimer formation 235 8.5 Fluorescence polarization method 237 8.5.1 Choice of probes 237 8.5.2 Homogeneous isotropic media 240 8.5.3 Ordered systems 242 8.5.4 Practical aspects 242 8.6 Concluding remarks 245 8.7 Bibliography 245 9 Resonance energy transfer and its applications 247 9.1 Introduction 247 9.2 Determination of distances at a supramolecular level using RET 249 9.2.1 Single distance between donor and acceptor 249 9.2.2 Distributions of distances in donor–acceptor pairs 254 9.3 RET in ensembles of donors and acceptors 256 9.3.1 RET in three dimensions. Effect of viscosity 256 9.3.2 Effects of dimensionality on RET 260 9.3.3 Effects of restricted geometries on RET 261 9.4 RET between like molecules. Excitation energy migration in assemblies of chromophores 264 9.4.1 RET within a pair of like chromophores 264 9.4.2 RET in assemblies of like chromophores 265 9.4.3 Lack of energy transfer upon excitation at the red-edge of the absorption spectrum (Weber’s red-edge effect) 265 9.5 Overview of qualitative and quantitative applications of RET 268 9.6 Bibliography 271 10 Fluorescent molecular sensors of ions and molecules 273 10.1 Fundamental aspects 273 10.2 pH sensing by means of fluorescent indicators 276 10.2.1 Principles 276 10.2.2 The main fluorescent pH indicators 283 10.2.2.1 Coumarins 283 10.2.2.2 Pyranine 283 10.2.2.3 Fluorescein and its derivatives 283 10.2.2.4 SNARF and SNAFL 284 Contents ix 10.2.2.5 PET (photoinduced electron transfer) pH indicators 286 10.3 Fluorescent molecular sensors of cations 287 10.3.1 General aspects 287 10.3.2 PET (photoinduced electron transfer) cation sensors 292 10.3.2.1 Principles 292 10.3.2.2 Crown-containing PET sensors 293 10.3.2.3 Cryptand-based PET sensors 294 10.3.2.4 Podand-based and chelating PET sensors 294 10.3.2.5 Calixarene-based PET sensors 295 10.3.2.6 PET sensors involving excimer formation 296 10.3.2.7 Examples of PET sensors involving energy transfer 298 10.3.3 Fluorescent PCT (photoinduced charge transfer) cation sensors 298 10.3.3.1 Principles 298 10.3.3.2 PCT sensors in which the bound cation interacts with an electron- donating group 299 10.3.3.3 PCT sensors in which the bound cation interacts with an electron- withdrawing group 305 10.3.4 Excimer-based cation sensors 308 10.3.5 Miscellaneous 310 10.3.5.1 Oxyquinoline-based cation sensors 310 10.3.5.2 Further calixarene-based fluorescent sensors 313 10.3.6 Concluding remarks 314 10.4 Fluorescent molecular sensors of anions 315 10.4.1 Anion sensors based on collisional quenching 315 10.4.2 Anion sensors containing an anion receptor 317 10.5 Fluorescent molecular sensors of neutral molecules and surfactants 322 10.5.1 Cyclodextrin-based fluorescent sensors 323 10.5.2 Boronic acid-based fluorescent sensors 329 10.5.3 Porphyrin-based fluorescent sensors 329 10.6 Towards fluorescence-based chemical sensing devices 333 Appendix A. Spectrophotometric and spectrofluorometric pH titrations 337 Appendix B. Determination of the stoichiometry and stability constant of metal complexes from spectrophotometric or spectrofluorometric titrations 339 10.7 Bibliography 348 11 Advanced techniques in fluorescence spectroscopy 351 11.1 Time-resolved fluorescence in the femtosecond time range: fluorescence up-conversion technique 351 11.2 Advanced fluorescence microscopy 353 11.2.1 Improvements in conventional fluorescence microscopy 353 11.2.1.1 Confocal fluorescence microscopy 354 11.2.1.2 Two-photon excitation fluorescence microscopy 355 11.2.1.3 Near-field scanning optical microscopy (NSOM) 356 x Contents [...]... 383 xi Molecular Fluorescence: Principles and Applications Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic) xii Preface 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. .. Interscience, New York Pringsheim P and Vogel M (1943) Luminescence of Liquids and Solids and its Practical Applications, Interscience, New York Monographs and edited books after 1960 Baeyens W R G, de Keukeleire D and Korkidis K (Eds) (1991) Luminescence Techniques in Chemical and Biochemical Analysis, Marcel Dekker, New York Becker R S (1969) Theory and Interpretation of Fluorescence and Phosphorescence, Wiley... Pioneering Contributions of Jean and Francis Perrin to Molecular Fluorescence, in: Valeur B and Brochon J C (Eds), New Trends in Fluorescence Spectroscopy Applications to Chemical and Life Sciences, Springer-Verlag, Berlin, pp 7–33 without emission of fluorescence), intersystem crossing (possibly followed by emission of phosphorescence), intramolecular charge transfer and conformational change These... (1968) Luminescence in Chemistry, Van Nostrand, London Chen R F and Edelhoch H (Eds) (1975, 1976) Biochemical Fluorescence Concepts, Vols 1 and 2, Marcel Dekker, New York Cundall R B and Dale R E (Eds) (1983) Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology, Plenum Press, New York Czarnik A W (Ed.) (1992) Fluorescence Chemosensors for Ion and Molecule Recognition, American Chemical... stay in his laboratory as a postdoctoral fellow; during this wonderful experience, I met outstanding scientists and friends like Dave Jameson, Bill Mantulin, Enrico Gratton and many others It is a privilege for me to belong to Weber’s ‘family’ Paris, May 2001 Bernard Valeur Molecular Fluorescence: Principles and Applications Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover);... (1946) Fluorescence et Phosphorescence, Hermann, Paris Dake H C and De Ment J (1941) Fluorescent Light and its Applications, Chemical Publishing Co., New York De Ment J (1945) Fluorochemistry A comprehensive Study Embracing the Theory and Applications of Luminescence and Radiation in Physicochemical Science, Chemical Publishing Co., New York ¨ Forster T (1951) Fluoreszenz organischer Verbindungen, Vandenhoeck... order parameters and molecular mobility can be locally evaluated by means of fluorescent probes Chapter 9 is devoted to resonance energy transfer and its applications in the cases of donor–acceptor pairs, assemblies of donor and acceptor, and assemblies of like fluorophores In particular, the use of resonance energy transfer as a ‘spectroscopic ruler’, i.e for the estimation of distances and distance distributions,... 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, solutions 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... lose the light’’, were already well aware of this.] Molecular Fluorescence: Principles and Applications Bernard Valeur > 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29919-X (Hardcover); 3-527-60024-8 (Electronic) 3 1 Introduction From the discovery of the fluorescence of Lignum Nephriticum (1965) to fluorescence probing of the structure and dynamics of matter and living systems at a molcular level ex arte... of a dye 1918 1919 1920 E L Nichols and E Merrit E L Nichols and E Merrit J Perrin Stern and Volmer F Weigert 1922 S J Vavilov 1923 1924 1924 S J Vavilov and W L Levshin S J Vavilov F Perrin 1924 F Perrin 1925 1925 1925 F Perrin W L Levshin J Perrin 1926 E Gaviola 1926 F Perrin 1927 E Gaviola and P Pringsheim E Jette and W West F Perrin 1928 1929 1929 1932 J Perrin and Choucroun F Perrin 1934 1935 1944 . Bernard Valeur Molecular Fluorescence Principles and Applications Molecular Fluorescence: Principles and Applications. Bernard Valeur > 2001 Wiley-VCH Verlag. (Electronic) Bernard Valeur Molecular Fluorescence Principles and Applications Weinheim – New York – Chichester – Brisbane – Singapore – Toronto Molecular Fluorescence: Principles and Applications. Bernard. 3-527-29965-3 Zander, C.; Keller, R. A. and Enderlein, J. Single-Molecule Detection in Solution. Methods and Applications 2002. isbn 3-527-40310-8 Molecular Fluorescence: Principles and Applications.