Molecular Fluorescence E-Book

Bernard Valeur

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 820

Veröffentlichungsjahr: 2013

Beschreibung

Molecular Fluorescence

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.

Leseprobe

Table of Contents

Further Titles of Interest

Title page

Copyright page

Preface to the First Edition

Preface to the Second Edition

Acknowledgments

Prologue

1 Introduction

1.1 What Is Luminescence?

1.2 A Brief History of Fluorescence and Phosphorescence

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

1.4 Various De-Excitation Processes of Excited Molecules

1.5 Fluorescent Probes, Indicators, Labels, and Tracers

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

Part I: Principles

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

2.1 Electronic Transitions

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

2.3 Selection Rules

2.4 The Franck–Condon Principle

2.5 Multiphoton Absorption and Harmonic Generation

3 Characteristics of Fluorescence Emission

3.1 Radiative and Nonradiative Transitions between Electronic States

3.2 Lifetimes and Quantum Yields

3.3 Emission and Excitation Spectra

4 Structural Effects on Fluorescence Emission

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

4.2 Fluorescence of Conjugated Polymers (CPs)

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

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

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

5 Environmental Effects on Fluorescence Emission

5.1 Homogeneous and Inhomogeneous Band Broadening – Red-Edge Effects

5.2 General Considerations on Solvent Effects

5.3 Solvent Relaxation Subsequent to Photoinduced Charge Transfer (PCT)

5.4 Theory of Solvatochromic Shifts

5.5 Effects of Specific Interactions

5.6 Empirical Scales of Solvent Polarity

5.7 Viscosity Effects

5.8 Fluorescence in Solid Matrices at Low Temperature

5.9 Fluorescence in Gas Phase: Supersonic Jets

6 Effects of Intermolecular Photophysical Processes on Fluorescence Emission

6.1 Introduction

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

6.3 Photoinduced Electron Transfer

6.4 Formation of Excimers and Exciplexes

6.5 Photoinduced Proton Transfer

7 Fluorescence Polarization: Emission Anisotropy

7.1 Polarized Light and Photoselection of Absorbing Molecules

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

7.3 Instantaneous and Steady-State Anisotropy

7.4 Additivity Law of Anisotropy

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

7.6 Case of Motionless Molecules with Random Orientation

7.7 Effect of Rotational Motion

7.8 Applications

8 Excitation Energy Transfer

8.1 Introduction

8.2 Distinction between Radiative and Nonradiative Transfer

8.3 Radiative Energy Transfer

8.4 Nonradiative Energy Transfer

8.5 Determination of Distances at a Supramolecular Level Using FRET

8.6 FRET in Ensembles of Donors and Acceptors

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

8.8 Overview of Qualitative and Quantitative Applications of FRET

Part II: Techniques

9 Steady-State Spectrofluorometry

9.1 Operating Principles of a Spectrofluorometer

9.2 Correction of Excitation Spectra

9.3 Correction of Emission Spectra

9.4 Measurement of Fluorescence Quantum Yields

9.5 Possible Artifacts in Spectrofluorometry

9.6 Measurement of Steady-State Emission Anisotropy: Polarization Spectra

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

10 Time-Resolved Fluorescence Techniques

10.1 Basic Equations of Pulse and Phase-Modulation Fluorimetries

10.2 Pulse Fluorimetry

10.3 Phase-Modulation Fluorimetry

10.4 Artifacts in Time-Resolved Fluorimetry

10.5 Data Analysis

10.6 Lifetime Standards

10.7 Time-Resolved Polarization Measurements

10.8 Time-Resolved Fluorescence Spectra

10.9 Lifetime-Based Decomposition of Spectra

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

11 Fluorescence Microscopy

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

11.2 Super-Resolution (Subdiffraction) Techniques

11.3 Fluorescence Lifetime Imaging Microscopy (FLIM)

11.4 Applications

12 Fluorescence Correlation Spectroscopy and Single-Molecule Fluorescence Spectroscopy

12.1 Fluorescence Correlation Spectroscopy (FCS)

12.2 Single-Molecule Fluorescence Spectroscopy

Part III: Applications

13 Evaluation of Local Physical Parameters by Means of Fluorescent Probes

13.1 Fluorescent Probes for Polarity

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

13.3 Temperature

13.4 Pressure

14 Chemical Sensing via Fluorescence

14.1 Introduction

14.2 Various Approaches of Fluorescence Sensing

14.3 Fluorescent pH Indicators

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

14.5 Fluorescent Molecular Sensors of Metal Ions

14.6 Fluorescent Molecular Sensors of Anions

14.7 Fluorescent Molecular Sensors of Neutral Molecules

14.8 Fluorescence Sensing of Gases

14.9 Sensing Devices

14.10 Remote Sensing by Fluorescence LIDAR

Appendix 14.A. Spectrophotometric and Spectrofluorometric pH Titrations

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

15 Autofluorescence and Fluorescence Labeling in Biology and Medicine

15.1 Introduction

15.2 Natural (Intrinsic) Chromophores and Fluorophores

15.3 Fluorescent Proteins (FPs)

15.4 Fluorescent Small Molecules

15.5 Quantum Dots and Other Luminescent Nanoparticles

15.6 Conclusion

16 Miscellaneous Applications

16.1 Fluorescent Whitening Agents

16.2 Fluorescent Nondestructive Testing

16.3 Food Science

16.4 Forensics

16.5 Counterfeit Detection

16.6 Fluorescence in Art

Appendix: Characteristics of Fluorescent Organic Compounds

Epilogue

Index

Further Titles of Interest

Sauer, M., Hofkens, J., Enderlein, J.

Handbook of Fluorescence Spectroscopy and Imaging

From Single Molecules to Ensembles

2011

ISBN: 978-3-527-31669-4

Goldys, E. M.

Fluorescence Applications in Biotechnology and Life Sciences

2009

ISBN: 978-0-470-08370-3

Strehmel, B., Strehmel, V., Malpert, J. H.

Applied and Industrial Photochemistry

2013

ISBN: 978-3-527-32668-6

Likhtenshtein, G.

Solar Energy Conversion

Chemistry of Solar Cells and Other Photochemical Systems

2012

ISBN: 978-3-527-32874-1

Ronda, C. R. (Ed.)

Luminescence

From Theory to Applications

2008

ISBN: 978-3-527-31402-7

Schlücker, S. (Ed.)

Surface Enhanced Raman Spectroscopy

Analytical, Biophysical and Life Science Applications

2011

ISBN: 978-3-527-32567-2

The Authors

Prof. Dr. Bernard Valeur

Conservatoire National des Arts et Métiers

292 rue Saint-Martin

75003 Paris

France

Prof. Mário Nuno Berberan-Santos

Centro de Química-Física Molecular

Instituto Superior Técnico

Av. Rovisco Pais

1049-001 Lisboa

Portugal

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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 derivatization, formation of a fluorescent complex, or fluorescence quenching have been developed. Fluorescence sensing is the method of choice for the detection of analytes with a very high sensitivity, and often has an outstanding selectivity thanks to specially designed fluorescent molecular sensors. For example, clinical diagnosis 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, 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 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 fluorimetric 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 phenomena and/or the structural parameters of the system under study.

Progress in instrumentation has considerably improved the sensitivity of fluorescence 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 techniques, which is a prerequisite for its practical use. The parameters that may affect the characteristics of fluorescence emission are numerous. This is a source of richness but also of complexity. The literature is teeming with examples of erroneous 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 developments 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 Berberan-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 preparation 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 Mantulin, Enrico Gratton, and many others. It is a privilege for me to belong to Weber’s “family.”

Bernard ValeurParis, May 2001

Preface to the Second Edition

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 crystals, 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, environment, 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 applications. 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, fluorescence 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.

Bernard ValeurParis, November 2011

Acknowledgments

The authors wish to thank all their colleagues who participated in fruitful discussions 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.

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

… 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; …]

Licetus, 1640 (about the Bologna stone)

1.1 What Is Luminescence?

The word luminescence, which comes from the Latin (lumen = light) was first introduced as luminescenz by the physicist and science historian Eilhardt Wiedemann in 1888, to describe “all those phenomena of light which are not solely conditioned by the rise in temperature,” as opposed to incandescence. Luminescence is often considered as cold light whereas incandescence is hot light.

Luminescence is more precisely defined as follows: spontaneous emission of radiation from an electronically excited species or from a vibrationally excited species not in thermal equilibrium with its environment.1) The various types of luminescence are classified according to the mode of excitation (see Table 1.1).

Table 1.1 The various types of luminescence.

Phenomenon

Mode of excitation

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

Luminescent compounds can be of very different kinds:

Organic compounds

: aromatic hydrocarbons (naphthalene, anthracene, phenanthrene, 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 (), lanthanide ions (e.g., Eu

3+

, Tb

3+

), doped glasses (e.g., with Nd, Mn, Ce, Sn, Cu, Ag), crystals (ZnS, CdS, ZnSe, CdSe, GaS, GaP, Al

2

O

3

/Cr

3+

(ruby)), semiconductor nanocrystals (e.g., CdSe), metal clusters, carbon nanotubes and some fullerenes, etc.

Organometallic compounds

: porphyrin metal complexes, ruthenium complexes (e.g., ), copper complexes, complexes with lanthanide ions, com­plexes 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.

Figure 1.1 Position of photoluminescence in the frame of light–matter interactions.

1.2 A Brief History of Fluorescence and Phosphorescence

It is worth giving a brief account of the history of fluorescence and phosphorescence. 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 scientists. The story of fluorescence started with a report by N. Monardes in 1565, but scientists focused their attention on light emission phenomena other than incandescence only in the nineteenth century. However, the major experimental and theoretical aspects of fluorescence and phosphorescence were really understood 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.

Table 1.2 Milestones in the history of fluorescence and phosphorescencea).

Year

Scientist

Observation or achievement

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