Time-resolved fluorescence principles

Mathis G, Socquet F, Viguier M, Darbouret B (1997) Homogeneous immunoassays using rare earth cryptates and time resolved fluorescence principles and specific advantages for tumor markers. Anticancer Res 17 3011-3014... 

Morgan, C. G., Mitchell, A. C. and Murray, J. G. (1990). Nano-second time-resolved fluorescence microscopy principles and practice. Trans. R. Microsc. Soc. 1, 463-6. 

The principle of the determination of time-resolved fluorescence spectra is described in Section 6.2.8. For solvent relaxation in the nanosecond time range, the single-photon timing technique can be used. The first investigation using this technique was reported by Ware and coworkers (1971). Figure 7.3 shows the reconstructed spectra of 4-aminophthalimide (4-AP) at various times after excitation. 

E. P. Diamindis, Immunoassays with time-resolved fluorescence spectroscopy Principles and applications, Clin. Biochem. 21, 139-150(1988). 

Volume 4 is intended to summarize the principles required for these biomedical applications of time-resolved fluorescence spectroscopy. For this reason, many of the chapters describe the development of red/NIR probes and the mechanisms by which analytes interact with the probes and produce spectral changes. Other chapters describe the unique opportunities of red/NIR fluorescence and the types of instruments suitable for such measurements. Also included is a description of the principles of chemical sensing based on lifetimes, and an overview of the ever-important topic of immunoassays. 

Basic principles and applications of time-resolved fluorescence spectroscopy have been outlined in a very illustrative way by Valeur [16]. Although punctiform spectroscopy is still the best way to get a detailed knowledge of all the important parameters that characterize fluorescence emission (exact spectral properties, decay time behavior, polarization), imaging is always preferred whenever the localization of the distribution of any biomolecule of interest is required or a great number of samples have to be analyzed [22]. 

Requirements with respect to the label used to mark one of the immunoreagents are comparable to those in other postcolumn reaction detection systems [4]. The label should preferably allow sensitive and rapid detection and be nontoxic, stable, and commercially available. So far, mainly fluorescence labels have been employed (e.g., fluorescein), although, in principle, also liposomes, time-resolved fluorescence, and electrochemical or enzymatic labels are feasible. On the other hand, labels providing a slow response, including radioactive isotopes and glow-type chemiluminescence, are less suitable for immunodetection. 

Diaraandis E. Immunoassays with time resolved fluorescence spectroscopy Principles and applications. 

While useful as a simple, model case with which one can demonstrate many of the principles involved in vibrational coherence, IVR between two vibrational levels is clearly not a very general situation. In view of the expectation that vibrational coupling in molecules may involve any number of levels and that many such cases of multilevel IVR are amenable to study by picosecond spectroscopy, it is useful to have theoretical guidelines301 pertaining to the manifestations that a system of N coupled vibrational levels might exhibit in time-resolved fluorescence. Hence, we consider now the situation depicted in Fig. 2. 

The bimolecular reaction dynamics of geminate recombination or acid-base neutralization have until recently been studied with time-resolved techniques probing electronic transitions. Time-resolved fluorescence using time-correlated single photon counting detection is limited to a time resolution of a few picoseconds. UV/vis pump-probe experiments, in principle, may have a time resolution of a few tens of femtoseconds, but may be hampered by overlapping contributions of... 

Steady-state and time-resolved fluorescence spectroscopy Absorption and fluorescence spectra were measured with a Hitachi 557 spectrophotometer and a Hitachi 850 spectrofluorometer, respectively. The time-resolved fluorescence spectra were measured with the apparatus reported previously [4,6] in principle, the time-correlated single photon counting system under a low excitation condition. The pulse intensity (540 nm, 6 ps (fwhm)) was in a range of 10 to 10 photons/cm. The time resolution of our optical set-up was 6 ps. Correction of spectral sensitivity and data treatment were carried out as reported previously [4,6]. 

Finally, the ability to produce pulsed laser radiation with a short lifetime on the fluorescence timescale opens up the possibility of applying time-resolved detection principles to improve the signal-to-back-ground ratio (see Figure 5). 

Time-resolved fluorescence measurements are frequently used in conjunction with flash initiation (cf. Sections 6.2.3.3,6.2.3.4, 7.2.2), but the principle is then quite different from that of quenching fluorescence is used simply to monitor concentration changes after the flash. 

Abstract In the first part of this chapter we will illustrate circular dichroism and we will discuss the optical activity of chemical compounds with respect to light absorption which is at the basis of this technique. Moreover, we will introduce the phenomena that lie behind the technique of optical rotatory dispersion. We thought appropriate to include a brief description of linear dichroism spectroscopy, although this technique has nothing to do with optical activity. In the final part of the chapter we will introduce the basic principles of the luminescence teehniques based on polarized (either circularly or linearly) excitation. The experimental approach to the determination of steady-state and time resolved fluorescence anisotropy will be illustrated. For all the teehniques examined in this chapter the required instrumentation will be schematieally deseribed. A few examples of application of these techniques to molecular and supramolecular systems will also be presented. 

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