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New Methods in Fluorescence Spectroscopy

Frontmatter

1. Fluorescence Spectroscopy: Where We Are and Where We’re Going

Abstract
Fluorescence, phosphorescence and chemiluminescence spectroscopies have established themselves as safe and ultrasensitive analytical techniques. However, the luminescence spectroscopies of today have moved well beyond the simple lamp, monochromator, sample compartment and detector arrangements of three or four decades ago, to very sophisticated electronic and optical instruments and some very elegant chemical and biochemical techniques and have drawn heavily on some of the most sophisticated physical and chemical theory for their current status. In this paper will be described some of the instrumentation and chemistry that have contributed to making luminescence spectroscopy one of the most powerful weapons in the chemist’s and biochemist’s arsenal and some of those approaches that will lead the way of this subject into the near future.
Steven G. Schulman

2. Interactions and Kinetics of Single Molecules as Observed by Fluorescence Correlation Spectroscopy

Abstract
Random fluctuations of the intensity of individual molecules excited to fluorescence by a stationary light source provide information on important molecular properties such as rotational motion [1–5], translational diffusion [6, 7], chemical kinetics [7–9] as well as the lifetime of the excited state [1, 2, 10].
Rudolf Rigler, Jerker Widengren, Ülo Mets

3. Fast Optical Imaging Techniques

Abstract
The distinction between detectors for imaging and spectroscopy is becoming increasingly blurred. The use of two-dimensional detectors to allow the recording of many spectra simultaneously is rapidly becoming an essential part of many experimental set-ups where the requirement is to look at temporal or spatial spectral variations within one experiment. In many applications there is a need for good time resolution. Traditional systems are used that operate at the rate of 50 or 60 Hz commonly used by standard TV cameras. This paper will look at the limitations that are encountered in trying to take fluorescent images at much higher repetition (frame) rates.
Craig D. MacKay

4. Kinetic Studies on Fluorescence Probes Using Synchrotron Radiation

Abstract
Many flexible aromatic molecules undergo spontaneous intramolecular rotational relaxation processes in the excited state leading to an energy minimum far away from the initial geometry which thus can be termed a photochemical product. Because the process occurs entirely in the excited state, it is called an “adiabatic photoreaction” [1]. An experimentally very well-studied example is the double-bond twisting of excited stilbene [2]. In recent years, the family of the Twisted Intramolecular Charge Transfer (TICT) compounds has been developed to a great extent [3–6]. In these compounds, two aromatic moieties are linked by a single bond, and excited-state rotational relaxation occurs towards a twisted conformation, coupled with intramolecular electron transfer (Fig. 4.1). Modern theoretical concepts can describe the twisting of both double and single bonds in one and the same model, that of biradicaloid states [6–8].
Wolfgang Rettig

5. Dynamics and Geometry in Dimeric Flavoproteins from Fluorescence Relaxation Spectroscopy

Abstract
The biologically widespread group of flavoproteins have in common that they contain the yellow flavin molecule as prosthetic group. The most common natural flavins are flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) which have both riboflavin as their biological precursor. To the biological chemist the most interesting feature of this versatile molecule is its redox properties which can be modulated by the (protein) environment. The redox active part of the natural flavins is the isoalloxazinic ring which can exist in the oxidized, one-electron reduced and two-electron reduced state. The redox potentials of the two one-electron steps vary greatly among different flavoproteins and depend on the chemical nature of the active site in which the isoalloxazine resides. This property makes flavin suitable as an electron shuttle in very different chemical (redox) reactions which explains its widespread occurrence in nature [1].
Philippe I. H. Bastiaens, Antonie J. W. G. Visser

6. Fluorescence Spectroscopy on Light Scattering Materials

Abstract
Fluorescence and light scattering are independent processes that appear simultaneously in many systems of biological, analytical, and technical interest (e.g. in plants, tissues, polycrystalline pigments, microscopic preparations, printing products with optical brighteners, polymer blends, thin layer chromatograms, heterogeneous absorbents). In the following, some important consequences of multiple scattering on fluorescence will be discussed, considering especially the spatial distribution of the detectable radiation, fluorescence reabsorption and reemission, samples with gradients in the fluorophore concentration inclusive of microscopic structures, distortion of diffuse reflectance spectra by fluorescence, and finally chemical modifications of the fluorophores by interaction with the phase boundaries of micro-heterogeneous media.
Dieter Oelkrug, Ulrike Mammel, Manfred Brun, Reiner Günther, Stefan Uhl

7. Optical Sensors Based on Fluorescence Quenching

Abstract
A sensor is a device capable of continuously monitoring a physical parameter or the concentration of an analyte. Among the various types of sensors, electrochemical and optical sensors form the two largest groups. Optical sensors (“optrodes” or “optodes”) are mainly based on the detection of changes in absorbance, reflectance, fluorescence or chemiluminescence. But also Raman-scattering, refractive index, light polarization, light scattering and other optical properties have been used as analytical parameters.
Wolfgang Trettnak

New Applications of Fluorometry

Frontmatter

8. Fluorescence in Forest Decline Studies

Abstract
Plants have to cope with various stress factors such as environmental pollutants, mineral deficiencies, climate or parasite infection giving rise to the current phenomenon of “forest decline”. To assess the various impacts of these stress factors, complex biochemical procedures are mostly utilized demanding damage to the specimen and fairly high sample concentrations. In contrast to these, spectroscopic methods offer many advantages. They are fast, non-invasive, easy to perform, highly selective for specific plant pigments, and require only small samples.
H. Schneckenburger, W. Schmidt

9. Fluorescence Microscopy Studies of Structure Formation in Surfactant Monolayers

Abstract
Monolayers of water-insoluble surfactants (Langmuir monolayers) are important model system in basic research for studying the self-organization of organic molecules into two-dimensional layers [1, 2]. These floating films are also the basis for the build-up of complex multilayered structures on solid substrates, so-called Langmuir—Blodgett films (LB-films), which are of considerable interest in fundamental science and promise some future practical applications [3].
Hans Riegler, Helmuth Möhwald

10. Fluorescence Lifetime Imaging and Application to Ca2+ Imaging

Abstract
Fluorescence spectroscopy is widely utilized for research in the biosciences [1–8]. These applications have been focused on two divergent disciplines, time-resolved fluorescence and fluorescence microscopy. In time-resolved measurements one takes advantage of the high information content of the time-dependent decays to uncover details about the structure and dynamics of macromolecules [4]. Such measurements are performed almost exclusively using ps laser sources coupled with high speed “single-pixel” photodetectors. While some parallel measurements have been reported, these have been for a linear array detector providing wavelength rather than spatial resolution [9]. In contrast, fluorescence microscopy is most often used to determine the localization (intensity) of the species of interest, usually of proteins or other macromolecules [6, 7]. The acquisition of two-dimensional (2D) fluorescence images is preferentially accomplished with low-speed accumulating detectors [10], which are not capable of quantifying ps-ns fluorescence decays. Consequently, the high information content of time-resolved fluorescence is not usually available for studies of microscopic biological samples. This is particularly disadvantageous when one considers the sensitivity of fluorescence decay times to chemical and environmental factors of interest, such as local pH, cation concentration, oxygen, and polarity, to name a few.
Joseph R. Lakowicz, Henryk Szmacinski, Kazimierz Nowaczyk, Klaus W. Berndt, Michael L. Johnson

11. Ether Phospholipids in Membranes: Applications of Phase and Steady-State Fluorometry

Abstract
Almost all procaryotic and eucaryotic cellular membranes contain diacyl glycerophospholipids (Fig. 11.1) as their predominant bilayer components. Most animal cells exhibit in addition, large proportions of ether lipids in their membranes [1]. They belong predominantely to the plasmalogen type. They are alkenylacylglycerophospholipids in which the hydrophobic alkyl chain is linked to position 1 of glycerol via an enolether bond (Fig. 11.1). Increasing evidence has already been accumulated that alkenylacyl and diacyl glycerophospholipids exhibit different properties in artificial as well as in natural bilayer systems [2, 3].
A. Hermetter, E. Prenner, J. Loidl, E. Kalb, A. Sommer, F. Paltauf

12. Pyrene-Labelled Lipids as Fluorescent Probes in Studies on Biomembranes and Membrane Models

Abstract
The immense progress in the understanding of the functions of proteins and nucleic acids in living cells is still contrasted by the limited comprehension of the significance of lipids and their structural diversity in the self-assembly and functionalization of biological membranes. Nevertheless, taking into account the costly maintenance of the specific tissue, cell, and cell organelle lipid compositions and their alterations it is obvious that the individual lipid classes do serve more important roles than what is at present commonly accepted [1].
Paavo K. J. Kinnunen, Anu Koiv, Pekka Mustonen

13. Optical Detection of Intracellular Ion Concentrations

Abstract
Fluorescence techniques give better results in biological and medical applications than do absorption techniques because of their higher sensitivity (the absorption signal is related to the 100% incident light intensity, while in fluorescence small signals are detected against zero background = darkness) and easier separation of the dye-related signal from the background.
Jan Slavík

Fluorimetric Analysis

Frontmatter

14. Analytical Applications of Very Near-IR Fluorimetry

Abstract
Fluorescence spectrometry has been accepted for many years as a major technique for trace analysis, and it is applied routinely and successfully to such diverse fields as the detection of solutes in flowing systems (e.g. in HPLC, CZE, and FIA); the monitoring of biospecific reactions, as in immunoassays and DNA-probe and enzyme assays; the detection of inorganic ions such as H+ and Ca2+ in cellular and other samples; the detection of components separated by laminar methods such as TLC and zone electrophoresis; and the study of molecular interactions, such as ligand-protein binding. It is noteworthy that in most of these assays it is not the intrinsic fluorescence of the determinant which is measured (polynuclear aromatic hydrocarbons are the major exception to this generalization): in most applications fluorescent labels or probes are used to provide the desired optical and molecular properties. [In this paper the term label is used to describe a covalently bound fluorophore group, while probe means a fluorophore, usually non-covalently bound to a protein or other surface, whose environment dependent fluorescence properties give information on the polarity of its binding sites]. The major advantages of fluorimetry in such fields are increased selectivity compared with UV-Visible absorption spectrometry, the great variety of sample handling methods available, and most of all the exceptional limits of detection accessible in favourable circumstances.
James N. Miller, Marc B. Brown, Nichola J. Seare, Stephen Summerfield

15. Fluorescence Detection in Flow Injection Analysis

Abstract
According to Taylor [1], a clear distinction should be made between analytical methods and techniques. In doing so, Flow Injection Analysis (FIA) can be regarded as composed of a firmly established series of continuous-flow methodologies developed for introduction of samples and standards into instruments (mainly optical and electrochemical) with or without a chemical derivatization reaction and/or continuous separation. The maturity of FIA is reflected in the large number of research papers (over 3000) and monographs [2–4] published on it, as well as on its steadily increasing use by routine and R&D laboratories. In short, FIA can be considered to be a useful analytical tool for solving a wide variety of analytical problems [5].
M. Valcárcel, M. D. Luque de Castro

16. Fluorescence Spectroscopy in Environmental and Hydrological Sciences

Abstract
One of man’s earliest scientific endeavours involved observations of fluorescence and phosphorescence effects which, over time, led to our present understanding of the underlying electron structure of matter. In the newer disciplines such as hydrology and environmental science the earliest application of fluorescence involved tracing the flow of surface-waters from point to point. This rather minimal use of the fluorescence effect was the only early major hydrologic application and even today remains a workhorse method. In the past, a drawback to the use of fluorescence data to produce more detailed results was the lack of specificity. This has not been true for the past fifteen years. Advances in light sources, light detection, electronics, and optics have resulted in techniques for adding njore specificity to fluorescence data. In much of the work discussed in this paper, excitation-emission matrix (EEM) patterns are utilized as pattern recognition techniques and as semi-quantitative techniques to follow the transport of natural and anthropogenic materials in hydrologic systems.
Marvin C. Goldberg, Eugene R. Weiner

Fluorescence Immunoassay

Frontmatter

17. Fluorescence Polarisation Immunoassay

Abstract
Since the introduction of radio immunoassay techniques [1], many different assay principles based on antibodies as specifiers have become routine tools in clinical diagnostics. Heterogeneous immunoassays, e.g. enzyme linked immunosorbant assays (ELISA) [2], require a series of time consuming incubation and wash steps, but they are characterized by a high sensitivity with a detection limit of 10−13 moll−1 and even lower.
Christian Klein, Hans-Georg Batz, Brigitte Draeger, Hans-Joachim Guder, Rupert Herrmann, Hans-Peter Josel, Ulrich Nägele, Roland Schenk, Bernd Vogt

18. Progress in Delayed Fluorescence Immunoassay

Abstract
The utilization of delayed detection of the emission in fluoroimmunoassays relates to the desire to improve the analytical sensitivity by means of temporal background rejection. Background is present in all fluorometric determinations and is due, among other things, to light scattering, emissions from the samples’ endogenous fluorescence, autofluorescence of cells and tissues, luminescent properties of solid matrixes, cuvettes, test tubes, lenses, etc. Time-resolved fluorometry with pulsed excitation can be used to eliminate the interfering background, provided that the decay time of the specific signal clearly differs from that of the background (Fig. 18.1). The efficient use of time-resolved detection requires the employment of delay times longer than 10 µs, and, accordingly, luminescent probes exhibiting excited state life times longer than 10 µs must be developed for the system.
Ilkka Hemmilä

19. Chemiluminescence Detection in Immunochemical Techniques. Applications to Environmental Monitoring

Abstract
Chemiluminescence is a phenomenon which has attracted increasing interest in recent literature not only because of its inherently great sensitivity, but also by the potentially widespread applications.
Eugène H. J. M. Jansen

Fluorescence in Biomedical Sciences

Frontmatter

20. Fluorescence Transients in Neurobiology: Applications of Voltage Sensitive and Ion Indicator Dyes

Abstract
The use of time resolved fluorescence techniques in physiology has experienced a resurgence in recent years as a consequence of the synthesis of new indicator dyes responsive to interesting physiological parameters and the improvement of sophisticated computer-based imaging devices. The combination of these two developments has allowed physiologists to follow specific biological events in individual cells with high spatial and temporal resolution. This gives a global view of physiological events instead of the parochial perspective usually achieved with single site recording devices.
William N. Ross

21. Optical Monitoring of Postsynaptic Potential in the Early Embryonic Avian Brain Stem Using a Voltage-Sensitive Dye

Abstract
The ontogenetic approach to generation of physiological events during natural development would be a useful and powerful strategy for studying central nervous systems: it would allow us to analyse progressively the complicated functional organization and architecture of nervous systems, in a manner reminiscent of the expansion of a complex function in a power series. However, the experimental analysis of early embryonic nervous systems is technically difficult because the cells are extremely inaccessible: the microelectrode examination of neural cells, which provides the most direct test of their electrophysiology, is often difficult because of the small size of the cells. For this reason, electrophysiological studies of very early developing embryonic nervous systems have been hampered.
K. Kamino, T. Sakai, Yoko Momose-Sato, H. Komuro, A. Hirota, K. Sato

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