Preface
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LUMINESCENCE YIELDS DATA
that often cannot be provided by any other methodology. This book is a compilation of a wide variety of original research contributions. Substantial information is given on the use of luminescence techniques to understand specific cell responses and the chemical mechanisms of cell action. An examination of natural environments is presented in the form of specific studies that characterize materials in both solid and liquid form and give information on the respective reactions of these materials in soil and water systems. Advanced research on standardization and standards developed for luminescence studies, as well as both active and passive use of luminescence, is included. Developments in laser design, microelectronics, and computers over the past 20 years have resulted in a renaissance for fundamental and analytical applications of molecular luminescence. Twenty years ago the conventional wisdom was that, although luminescence is inherently very sensitive for detection of emitters, the generally broad, featureless emission band and the frequent interferences from trace contaminants made interpretation of luminescence spectra difficult. It became necessary to isolate samples of unknown materials to such a high degree that luminescence measurements did not contribute much additional material. Today, new techniques have made luminescence spectroscopy indispensable as a sensor of chemical species in environments as diverse and complex as biochemical fluids, chromatographic columns, the Earth's atmosphere and waters. Furthermore, luminescence allows the determination of internal energy states and follows how they change with time on a scale that can be as short as one picosecond. Such measurements are crucial for fundamental investigations of the dynamics of chemical processes. For analytical applications, many techniques have greatly improved our ability to analyze multicomponent systems. These include the coupling of luminescent detectors with chromatographic columns and other separation devices; combining lifetime and polarization data with spectral measurement; three-dimensional plotting of the total excitation-emission intensity matrix; synchronous scanning of the excitation and emission wavelengths, tagging specific molecules in a mixture with unique fluorescent labels; and using tunable lasers to induce emission from electronic states that are characteristic of ix Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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particular molecules. Chemi-excitation of luminescent molecules is another versatile way to impart additional sensitivity and specificity to the analysis of multicomponent systems. Compounds such as amines and amino acids have been derivatized and made to react with oxalates and hydrogen peroxide after high-performance liquid chromatography separations. The chemiluminescent reactions permit detection at femtomole levels. Fluorescence analysis has been extended to many nonfluorescent species by the development of a wide range of derivatizing agents that form a fluorescent product. This approach has been especially useful with biochemical molecules, many of which are not natural fluorophores. Luminescence lifetimes are measured by analyzing the rate of emission decay after pulsed excitation or by analyzing the phase shift and demodulation of emission from chromophores excited by an amplitudemodulated light source. Improvements in this type of instrumentation now allow luminescence lifetimes to be routinely measured accurately to nanosecond resolution, and there are increasing reports of picosecond resolution. In addition, several individual lifetimes can be resolved from a mixture of chromophores, allowing identification of different components that might have almost identical absorption and emission features. In studies of molecular dynamics, lasers of very short pulse lengths allow investigation by laser-induced fluorescence of chemical processes that occur in a picosecond time frame. This time period is much less than the lifetimes of any transient species that could last long enough to yield a measurable vibrational spectrum. Such measurements go beyond simple detection and characterization of transient species. They yield details never before available of the time behavior of species in fast reactions, such as temporal and spatial redistribution of initially localized energy in excited molecules. Laser-induced fluorescence characterizes the molecular species that have formed, their internal energy distributions, and their lifetimes. Biological applications of luminescence make use of resonance energy transfer as a microscopic ruler to measure distances between chemical groups in complicated biological structures. Electronic excitation of a fluorophore can be dissipated either by fluorescent emission or by nonradiative resonant energy transfer to an acceptor molecule. The transfer rate depends on the distance between donor and acceptor groups. Fluorescence lifetimes are shortened as the fraction of energy dissipated nonradiatively increases. Resonant energy transfer is particularly significant over the distance range of 2 to 5 nm, which is also the range of typical protein diameters and membrane thicknesses. As a result, fluorescent lifetime measurements of natural and derivative fluorophores are used to determine distances between binding sites on χ
Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
proteins and chromophore distances in biological aggregates. Such measurements have produced important results regarding the conformation and structures of several proteins and geometric details of protein-membrane interactions. Particularly valuable is the fact that luminescence measurements of molecular conformation can often be made in situ, yielding unique information not possible from X-ray diffraction about molecules that change shape when crystallizing or do not form crystals at all. Many biologically important processes are related to changes in activity caused by environmental changes at metal binding sites on proteins. The normally bound ions Ca and Mg can be replaced by the transition metal ion fluorescent probes Tb and Eu , whose luminescent lifetimes are very sensitive to their chemical environment, especially the degree of hydration. Their emission lifetimes can be used to track environmental changes at binding sites while a protein is reacting or responding to external influences. There are many other instances where the sensitivity of chromophore lifetimes to their chemical surroundings has been used to probe the details of environmental conditions around a chromophore, such as solvation, micelle structures, and the structures of dissolved complexes. The reactivity of large and complex molecules is often closely related to their size and shape. Luminescence polarization sometimes offers a way to determine these properties. Electronic excitation of a chromophore with polarized light will produce polarized emission. However, any rotation of the molecule after excitation but before emission will cause some depolarization of the emission. Depolarization and lifetime measurements can be used together to determine rates of rotation of chromophoric molecules that can be related to a molecule's rotational diameter. Studying the temperature behavior of depolarization can yield additional information about a molecule's shape. Observing how rotational rates change under different conditions can indicate corresponding changes in the shape of a molecule, such as denaturation of proteins at high temperatures or complex formation at high concentrations. Luminescence science has developed into a powerful tool for studying nature in macro- and microenvironments. At present, there are many scientific advances being reported that incorporate fluorescence technology into the research regimen. I expect this trend to continue as the scientific community becomes more cognizant of the knowledge to be gained by use of luminescence techniques. The purpose of the symposium on which this book is based was to report original research advances that use luminescence as a basic investigative tool. Because these applications transcend the field of chemistry, a multidisciplinary group of prominent scientists contributed 2+
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to the symposium to enrich it with a variety of applications. Researchers in the biological, chemical, and physical sciences have been in the forefront of those employing luminescence methodology and have brought some innovative uses of luminescence to bear on their research. This book presents excellent research results in the biological, chemical, environmental, and hydrological sciences. Each chapter addresses an important application of luminescence and advances its particular subject discipline.
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Acknowledgments I wish to acknowledge the assistance of the many people who contributed to this publication. Patricia M. Negomir's help in organizing this book was greatly appreciated. John B. Weeks gave material assistance to the compilation of the book. Peter L. Martin did an excellent job of editing the chapters. Patricia A. Griffiths, Linda S. Britton, and their staff supplied typing assistance that was invaluable. Ranee Velopoldi contributed to the design of the dust jacket. Finally, I wish to thank all of the contributing authors for being patient while awaiting publication of their material and for doing such an outstanding job in their respective fields. MARVIN C. GOLDBERG
U.S. Geological Survey Box 25046, MS 424 Lakewood, CO 80225 September 26, 1988
xii Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
