Principles of Fluorescence Spectroscopy

Second Edition

 Preface

It has been 15 years since publication of the first edition of Principles of Fluorescence Spectroscopy. This first volume grew out of a graduate level course on fluorescence taught at the University of Maryland. The first edition was written during a transition period in the technology and applications of fluorescence spectroscopy. In 1983, time-resolved measurements were performed using methods which are primitive by today's standards. The dominant light source for time-resolved fluorescence were the nanosecond flash lamps, which provided relatively wide excitation pulses. Detection was accomplished with relatively slow response PMTs. In the case of phase-modulation fluorometry, the available instruments operated at one or two fixed light modulation frequencies, and thus provided limited information on complex time-resolved decays. Data analysis was also limited because of the lower information contained in the experimental data.

Much has changed since 1983. The dominant light sources are now picosecond dye lasers or femtosecond Titanium Sapphire lasers. In the case of phase-modulation fluorometry, frequency-domain now operates over a range of light modulation frequencies, allowing resolution of complex decays. The time resolution in both domains has been increased by the introduction of high speed microchannel plate photomultiplier tubes. Data analysis has become increasingly sophisticated not only because of the availability of more powerful computers, but also because of the availability of additional data and the increased resolution available using global analysis. These advanced experiments and analysis capabilities have been extended to provide resolution of complex anisotropy decays, conformational distributions, and complex quenching phenomenon.

Another important change since 1983, has been the extensive development of fluorescence probes. Early fluorescent probes were those derived from histochemical staining of cells, a limited number of lipid and conjugatable probes, and of course, intrinsic fluorescence from proteins. Today the menu of fluorescence probes has expanded many-fold. A wide variety of lipid and protein probes have been developed, and probes have become available with longer excitation and emission wavelengths. There has been extensive development of cation sensing probes for use in cellular imaging. The nanosecond barrier of dynamic fluorescence information has been broken by the introduction of long lifetime probes.

Another example of the rapid expansion of fluorescence is DNA sequencing technology. Prior to 1985, most DNA sequencing was performed using radioactive labels. Since that time sequencing has been accomplished almost exclusively using fluorescent probes. The fluorescence technology for DNA sequencing is advancing rapidly due to the goal of sequencing the human genome. And finally, who would have expected in 1983 that the gene for the green fluorescence protein could be introduced into cells with spontaneous folding and formation of the fully fluorescent protein.

Parts of this book were influenced by a course taught at the Center for Fluorescence Spectroscopy, which has been attended by individuals from throughout the world. However, the most important factor stimulating the Second Edition were the positive comments of individuals who found value in the First Edition. Many individuals commented on the value of explaining the basic concepts from their fundamental origins. This has become increasingly important as the number of practitioners has increased, without a significant increase in courses at the undergraduate or graduate level.

In the second edition of "Principles" I have attempted to maintain the emphasis on basics, while updating the examples to include more recent results from the literature. There is a new chapter providing an overview of extrinsic fluorophores. The discussion of time-resolved measurements has been expanded to two chapters. Quenching has also been expanded in two chapters. Energy transfer and anisotropy have each been expanded to three chapters. There is also a new chapter on fluorescence sensing. To enhance the usefulness of this book as a text book, most chapters are followed by a set of problems. Sections which describe advance topics are indicated as such, to allow these sections to be skipped in an introduction course. Glossaries are provided for commonly used acronyms and mathematical symbols. For those wanting additional information, the final appendix contains a list of recommended books which expand on various specialized topics.

In closing I wish to express my appreciation to the many individuals who have assisted me not only in preparation of the book but also in the intellectual developments in my laboratory. My special thanks goes to Ms. Mary Rosenfeld for her careful preparation of the text. Mary has cheerfully tolerated the copious typing and numerous revisions of all the chapters. I also thank the many individuals who have proof-read various chapters, and provided constructive suggestions. These individuals include Felix Castellano, Robert E. Dale, Jonathan Dattelbaum, Maurice Eftink, John Gilchrist, Zygmunt Gryczynski, Petr Herman, Gabor Laczko, Li Li, Harriet Lin, Zakir Murtaza, Leah Tolosa, Bogumil Zelent, and I apologize for any omissions.

I also give my special thanks to Dr. Ignacy Gryczynski and his wife, Krystyna Gryczynska. While I started to write this book, Ignacy said "just go and write, don't worry about the figures." Many of the excellent figures in this book were drawn by Krystyna, with the valuable suggestions of Ignacy. Without their dedicated efforts the book could not have been completed in any reasonable period of time. I also thank Ms. Suzy Rhinehart for providing a supportive family environment during preparation of this book. And finally, I thank the National Institutes of Health and the National Science Foundation for support of my laboratory.

J.R. Lakowicz, Baltimore
Center for Fluorescence Spectroscopy
June 15, 1998

Contents :

Most sections of this book describe basic aspects of fluorescence spectroscopy, and some sections describe more advanced topics. These sections are marked "Advanced Topics," which can be omitted in an introduction course on fluorescence. The advanced chapters on quenching (Chapter 9), anisotropy (Chapter 12), and energy transfer (Chapters 14 and 15) can be skipped in a first reading. Depending on the interest of the reader, chapters 18 to 22 can also be skipped.

1.	Introduction to Fluorescence

	1.1	Phenomena of Fluorescence	
	1.2	Jablonski Diagram	
	1.3	Characteristics of Fluorescence Emission	
			1.3.1.	Stokes' Shift	
			1.3.2.	Emission Spectra are Typically Independent
				of the Excitation Wavelength	
			1.3.3.	Exceptions to the Mirror Image Rule	
	1.4	Fluorescence Lifetimes and Quantum Yields	
			1.4.1.	Fluorescence Quenching	
			1.4.2.	Time Scale of Molecular Processes in Solution	
	1.5	Fluorescence Anisotropy	
	1.6	Resonance Energy Transfer	
	1.7.	Steady State and Time-Resolved Fluorescence	
			1.7.1.	Why Time-Resolved Measurements?	
	1.8.	Biochemical Fluorophores	
			1.8.1.	Fluorescent Indicators	
	1.9.	Molecular Information From Fluorescence	
			1.9.1.	Emission Spectra and the Stokes Shift	
			1.9.2.	Quenching of Fluorescence	
			1.9.3.	Fluorescence Polarization or Anisotropy	
			1.9.4.	Resonance Energy Transfer	
	1.10.	Fluorescence Sensing	
	1.11.	Summary	


2.	Instrumentation for Fluorescence Spectroscopy

	2.1.	Excitation and Emission Spectra	
	2.2.	Light Sources	
	2.3.	Monochromators	
	2.4.	Optical Filters	
	2.5.	Optical Filters and Signal Purity	
	2.6.	Photomultiplier Tubes	
	2.7.	Polarizers	
	2.8.	Corrected Excitation Spectra	
	2.9.	Corrected Emission Spectra	
	2.10.	Quantum Yield Standards	
	2.11.	Effects of Sample Geometry	
	2.12.	Common Errors in Sample Preparation	
	2.13.	Absorption of Light and Deviation from the Beer-Lambert Law	
	2.14.	Two-Photon and Multi-Photon Excitation	
	2.15.	Conclusions	


3.	Fluorophores

	3.1.	Intrinsic or Natural Fluorophores	
	3.2.	Extrinsic Fluorophores	
	3.3.	Red and Near Infrared (NIR) Dyes	
	3.4.	DNA Probes	
	3.5.	Chemical Sensing Probes	
	3.6.	Special Probes	
	3.7.	Fluorescent Proteins	
	3.8	Long Lifetime Probes	
	3.9.	Proteins as Sensors	
	3.10.	Conclusion	


4.	Time-Domain Lifetime Measurements

5.	Frequency-Domain Lifetime Measurements

6.	Solvent Effects on Emission Spectra

7.	Dynamics of Solvent and Spectral Relaxation

8.	Quenching of Fluorescence

9.	Advanced Topics in Fluorescence Quenching

10.	Fluorescence Anisotropy

11.	Time-Dependent Anisotropy Decays

12.	Advanced Anisotropy Concepts

13.	Energy Transfer

14.	Time-Resolved Energy Transfer and Conformational Distributions of Biopolymers

15.	Energy Transfer to Multiple Acceptors in One-, Two- or Three-Dimensions

16.	Protein Fluorescence

17.	Time-Resolved Protein Fluorescence

18.	Excited State Reactions

19.	Fluorescence Sensing

20.	Long Lifetime Metal-Ligand Complexes

21.	DNA Technology

22.	Phase Sensitive and Phase Resolved Emission Spectra


Appendix I - Corrected Emission Spectra	  

Appendix II - Fluorescent  Lifetime Standards	  

Appendix III - Additional Reading