Biological Sciences / Biochemistry Jameson INTRODUC TION TO “An essential contribution …” —Ken Jacobson, Kenan Distinguished Professor of Cell Biology and Physiology, University of North Carolina at Chapel Hill FLUORESCENCE “… a delight to read.” INTRODUCTION TO —Beniamino Barbieri, President, ISS Inc. “Highly recommended …” —Luis Bagatolli, Center for Biomembrane Physics, University of Southern Denmark “ … exquisite …” —Zygmunt (Karol) Gryczynski and Ignacy Gryczynski, University of North Texas Health Science Center “… truly a masterwork …” FLUORESCENCE —Jerson L. Silva, National Institute of Science and Technology for Structural Biology and Bioimaging, Universidade Federal do Rio de Janeiro An indispensable resource for any life scientist’s library, Introduction to Fluores- cence helps you acquire a sound understanding of basic fluorescence theory and practice. In color throughout, the book takes you through the history of important discoveries to the most current advances.
It introduces the fundamentals of the flu- orescence phenomenon and gives detailed examples of fluorescence applications in the molecular life sciences, including biochemistry, biophysics, clinical chemis- try and diagnostics, pharmaceutical science, and cell and molecular biology. K10301 6000 Broken Sound Parkway, NW Suite 300, Boca Raton, FL 33487 ISBN: 978-1-4398-0604-3 711 Third Avenue New York, NY 10017 90000 an informa business 2 Park Square, Milton Park www.com Abingdon, Oxon OX14 4RN, UK 9 781439 806043 w w w. Jameson K10301 mech-rev3.indd 1 12/16/13 9:29 PM IN T RO D UC TION TO FLUORESCENCE IN T RO D UC TION TO FLUORESCENCE David M. Jameson CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2014 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.
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Visit the Taylor & Francis Web site at http://www.com and the CRC Press Web site at http://www.com Dedicated to Gregorio Weber, who taught me that scientists should put doubt above belief Contents Preface. xvii CHAPTER 1 Introduction.1 What Is Fluorescence and How Is It Used?.1 A Nano-History of Fluorescence.10 Excited State Lifetime.10 And the Rest.12 General Fluorescence Texts.12 CHAPTER 2 Absorption of Light.13 Electromagnetic Radiation: Characterization.14 Relevant Wavelength Range.15 Absorption of Light by Molecules.16 Franck–Condon Principle.17 Effect of Conjugation on Absorption.18 Effects of Molecular Environment on Absorption.21 Beer–Lambert Law.21 Departures from Beer–Lambert Law.25 vii Contents CHAPTER 3 Instrumentation.44 Photon Counting versus Analog Detection.56 CHAPTER 4 Emission and Excitation Spectra.57 Why Do We Want Emission Spectra?.58 Correcting Emission Spectra.62 Correcting for Background.63 Correcting for Instrumental Parameters.65 Spectral Center of Mass.72 Advanced Scanning Methods.74 CHAPTER 5 Polarization and Anisotropy.77 Some Applications of Polarization/Anisotropy.90 Protein/Ligand Interactions.92 Fluorescence Polarization Immunoassay.95 Numerical Aperture Effects.97 Effect of Scattering.97 Rayleigh Scatter and Rayleigh Ghosts.97 viii Contents Scattering of the Fluorescence.98 Depolarization via FRET.99 CHAPTER 6 Time-Resolved Fluorescence.101 Excited State Lifetimes.110 Time Correlated Single Photon Counting.111 Frequency Domain Instrumentation.114 Time Domain versus Frequency Domain.115 Anisotropy Decay/Dynamic Polarization.129 CHAPTER 7 Quantum Yields and Quenching. 131 Determination of QYs.132 Kasha–Vavilov Rule.134 Collisional or Dynamic Quenching.140 Quenching and Membrane Systems.143 CHAPTER 8 Förster Resonance Energy Transfer.147 How Do We Determine the Efficiency of Energy Transfer (E)?.150 Steady-State Intensity.150 Time-Resolved Method: Decrease in the Lifetime of the Donor.151 The Orientation Factor.153 ix Contents How Do We Determine κ2?.153 Homo-Transfer of Electronic Excitation Energy.155 Examples of FRET Applications.160 CHAPTER 9 Brief Overview of Fluorescence Microscopy.163 Introduction of Fluorophores into Living Cells.163 Fluorescence Microscopy Approaches.165 Total Internal Reflection Fluorescence.168 Multi-Photon Excitation.172 Fluorescence Fluctuation Spectroscopy (FFS).172 Fluorescence Recovery after Photobleaching (FRAP).181 Fluorescence Lifetime Imaging Microscopy (FLIM).183 Super-Resolution Techniques.186 Single-Molecule Fluorescence.191 Where Do Fluorophores Come From?.191 How Does One Choose a Fluorophore?.197 Labeling Proteins In Vitro.206 Amine Reactive Probes.207 Thiol Reactive Probes.210 Separation of Labeled Protein from Unreacted Probe.211 Determination of the Extent of Labeling.213 x Contents Membrane Probes.216 Additional Comments on Membrane Systems.225 Nucleic Acid Probes and Nucleotide/Nucleoside Analogs.237 Voltage-Sensitive Dyes.248 CHAPTER 11 Intrinsic Protein Fluorescence.258 Electronic Energy Transfer in Proteins.261 Use of Site-Directed Mutagenesis.266 Protein Unfolding/Refolding.268 Intrinsic Fluorescence Spectra/Intensity.269 Protein–Protein and Protein–DNA Interactions.271 Phasors and Protein Fluorescence.274 Appendix: Rogue’s Gallery of Fluorescence Artifacts and Errors. 277 xi Preface Aloha! First and foremost, I wish to welcome you to Introduction to Fluorescence! I also want to remind you that this book is an introduction to fluorescence, not an advanced treatise.
The literature already abounds with advanced treatments of each and every topic covered in this book. But having said that, I also want to be clear that this book still requires effort on your part if you wish to acquire a useful understanding of basic fluorescence theory and practice. My former graduate advisor, Gregorio Weber (whom we shall have occasion to mention throughout this book, since his contributions to the field were vast), liked to quote Oscar Wilde, in particular the phrase “The truth is rarely pure and never simple” from the play The Importance of Being Earnest. So it is with fluorescence.
In an introductory text, I feel obliged to convey the general principles and to use examples that demonstrate these principles. There are exceptions to almost every “rule” proposed in these pages. For example, when I state that fluorescence results from the transition from the first electronic excited state to the ground state, someone may point out that azulene can emit directly from a higher electronic level. Or when I state that the quantum yield of a fluorophore is independent of the exciting wavelength, I may be reminded that tryptophan excited at 230 nm has a lower yield than it does when excited at 280 nm due to photoelectron ejection from the upper electronic level.
But in the vast majority of cases, the “rules” I propose hold. If readers of this book demonstrate a sustaining interest in fluorescence and continue to work and study in the field, I have no doubt they will learn of the exceptional cases on their own and may even discover new ones! I should add that clearly not every fluorescence topic or system is covered in this short book. A search of the word “fluorescence” on PubMed, for example, will pull up several hundred thousand articles, which cover hundreds or perhaps thou- sands of different systems. Hence, it is quite possible that the reader’s favorite fluorescence system is not mentioned.
Sorry! For examples of many aspects of fluorescence, I have drawn frequently from my own work, not because my research is particularly interesting (to anyone other than myself!), but rather because I am most familiar with it. I have also mentioned many contributions from Gregorio Weber—in these cases, though, not only am I familiar with his work, but it IS highly significant. I must acknowledge here my debt to those who helped me, either indirectly or directly, to write this book. It goes without saying that I owe—and will xiii Preface continue to owe—much to Gregorio Weber, from whom I learned so much about fluorescence and also about life.
He not only taught in the traditional sense, he also taught by example. He was the great scientific inspiration of my life and, to paraphrase a remark once made by Ludwig (Lenny) Brand (himself a legendary fluorescence practitioner), Gregorio Weber showed by example how scientists should interact with one another, namely with cour- tesy, generosity, good humor, and humility. During the past four decades, I have also been fortunate to have known and worked with many outstanding scientists from whom I have also learned. Foremost among these is Enrico Gratton, who has been a friend since the day, many years ago, that I picked him up at the airport in Champaign, Illinois, upon his arrival to begin a post- doctoral position in Weber’s lab.
Enrico is one of the truly outstanding sci- entists of his generation and his Laboratory for Fluorescence Dynamics, an NIH Research Resource Center, has trained large numbers of students, post- docs, and visiting researchers on advanced fluorescence methodologies. Other friends in Weber’s lab during my graduate student days were Bernard Valeur, Antonie Visser, Joseph Lakowicz, Gregory Reinhart, Parkson Chong, and Bill Mantulin, who all went on to make outstanding contributions to the fluores- cence field. Many of my own students, postdocs, and collaborators over the years have contributed to my work in the fluorescence field, and I am grate- ful for their collaborations. I am grateful to my long-term collaborators and friends Joseph Albanesi, Juan Brunet, and Michael Anson for the countless pleasant hours of discussions on myriad topics, including fluorescence.
I also want to remember here two former colleagues and friends who passed away while I was working on this book. They are John Eccleston, with whom I col- laborated on fluorescence projects for more than 30 years, and Robert Clegg, whom I had known for many years and with whom I shared a passion for his- tory. I miss them both. I want to thank the present members of my laboratory, postdoc Nicholas James and undergraduate Remy Minami, for reading some of the book in its early form and for offering valuable suggestions.
Postdoc Carissa Vetromile, in addition to extensive proofreading, took many of the spectra shown and helped make many of the figures, and I am very grateful for her efforts. I thank Marcin Bury, in my lab, for his excellent and patient photography and proof- reading and also for drawing most of the chemical structures in this book. Leonel Malacrida deserves special mention for providing Prodan spectra as well as numerous figures. Greg Reinhart also offered useful suggestions, especially on protein fluorescence.
I wish to thank the various funding agen- cies that have supported my research over the years, including the National Science Foundation, the National Institutes of Health, and the American Heart Association. I am also grateful for the financial support provided in recent years by Allergan, Inc. I also thank the University of Hawaii for provid- ing the intellectual environment that supported this project. I must acknowledge Taylor & Francis and in particular Luna Han for origi- nally suggesting that I write this book.
I also thank Luna for her patience throughout the process—especially as suggested deadlines passed without bearing fruit. I thank Beniamino Barbieri, president of ISS, Inc., who organized xiv Preface many fluorescence workshops over the years and invited me to lecture in them. Organizing and presenting these lectures gave me the opportunity to clarify my thoughts on many topics and the opportunity to discuss material with hundreds of students. I thank the many students who, over the years, endured my lectures on fluorescence.