Time-resolved luminescence principle

Principle of time-resolved luminescence measurement using lanthanide labels 190... 

Molecular photophysics, especially the use of steady-state and time-resolved luminescence spectroscopy, have many important applications and there has been a progressive emergence of a new field of analytical chemistry based on these principles. It has been known for many decades that the excited state properties of certain molecules are highly sensitive to the local environment but it is only recently that a concerted effort has been made to use this sensitivity in a practical way. The main approaches to employing variations in photophysical properties as an analytical tool can be divided into two areas namely, (i) development of luminescent probes that respond to changes in the environment and (ii) identification of molecular systems for which the emission... 

Different variations of time-resolved luminescence assays were patented in 1982 by Wieder [1], in 1983 by Soini and HemmUa [2], and in 1999 by Diamandis [3]. The Finnish company Wallac first commercialized the principle and introduced an assay reader for dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) in the beginning of the 1980s. The first (1982) DELFIA-based bioassay for diagnostic market was for Rubella antibodies, and it was the first sensitive nonradioisotope innnunoassay marking the beginning of a new era [4]. 

The optical setup for the time-resolved luminescence measurement is in principle simple and includes the following components ... 

The idea of the use of luminescent lanthanide chelates in bioanalysis can be traced back to the 1980s, when Hemmila (1985) reported a time-resolved fluorometric system using an Eu labeling reagent, which was commercially produced by Wallac Oy company. The system was intended for immunoassay and included the spectrometer for time-resolved measurements. The basic principle of the immunoassay system (DELFIA ) is shown in fig. 1. 

Figure 13.2 Principles of (a) time-resolved spectroscopy, (h) heterogeneous immunoassays, and (c) homogeneous immunoassays [1]. (Reproduced from J.C.G Bunzli and C. Piguet, Taking advantage of luminescent lanthanide ions, Chemical Society Reviews, 34, 1048—1077, 2005, by permission of The Royal Society of Chemistry.)...

FIGURE 14. Principle of the time-resolved (gated) luminescence assay. Reproduced with permission from Reference 2, Copyright 2005 the Royal Society of Chemistry... 

Principally, the pump and probe technique depicted in Fig. 1.21 is apphed in time-resolved transient absorption experiments. A pump beam, directed onto the sample, generates excited species or reactive intermediates such as free radicals. The formation and decay of these species can be monitored with the aid of an analyzing (probe) light beam that passes through the sample perpendicular to the direction of the pump beam. In principle, a set-up of this kind is also suitable for recording luminescence, if it is operated without the probe beam. 

Multiphoton or two-photon laser scanning microscopy is an alternative to confocal and time-resolved microscopy for bioimaging applications. The principle has been discussed in Lanthanides Luminescence Applications and concerns a two-photon excitation from the simultaneous absorption of two photons in a single quantized event. A bioprobe that normally absorbs ultraviolet light (Xex = 350 nm) can also be excited by two photons of NIR light, at 700 nm (the wavelength is twice that required for one-photon excitation). These two photons must interact simultaneously, which means in a very small lapse time. The instrumentation requires pulse lasers to provide sufficient power, as the photon density must... 

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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