Terbium, fluorescent decay

Glasses. Pearson and Peterson 98) studied extensively the fluorescent decay properties of terbium in Calibo base glass which has the composition CaO, 20 Li20, 10 B203, 70 mole per cent. All their data was taken using a stroboscopic method. 

Huffman (87) studied the transient emissions from terbium in a vinylic resin matrix. His compound was Tb tris-[4,4,4-trifluoro-l-(2-thienyl)-1,3-butaneodione] in polymethylmethacrylate. This may be conveniently abbreviated as TbTTA in PMMA. The compound EuTTA in PMM A had previously been reported by Wolff and Pressley (99) to give laser oscillation. Working with small fibers at 77°K, Huffman found distortions from the normal fluorescent decay curves when the optical pumping was large. He interprets this as evidence for stimulated emission. A comparison of these distorted decays with EuTTA in PMMA indicated a similar behavior, thus tending to substantiate his hypothesis. 

To a very large extent, most of the recent data on fluorescent decay times of the other trivalent ions (those beside terbium, neodymium, and europium) stems in some way from laser experiments. In this section some representative data on these are considered. 

Cha et al. (1999) used a variant of FRET called LRET for lanthanide-based fluorescence energy transfer. In this technique (Selvin, 1996) the donor is terbium or europium which, in fact, is luminescent. There are several advantages of this technique over regular FRET. It has been found that terbium emits isotropically, which means that the uncertainty due to the dipole orientation is decreased to a maximum error of 10%. This error can be decreased even further if the anisotropy of the acceptor is also known. The second advantage is that the fluorescence decay has a time constant of about 1.5 ms, making it easily measurable with conventional recording techniques. The third advantage is that the emission of terbium is peaked and one can find fluorophores that emit in between peaks. This means that the fluorescence of the acceptor can be measured with little or no contamination from the donor. In addition, as the acceptor has a fast decay, any measurement of the acceptor fluorescence with decays comparable to the donor will exclude any possible direct... 

In n-butanol as solvent at 293 K Tb(acac)3-3H20 undergoes intermolecular energy transfer to the complexes R(acac)3-3H20 (R = Pr, Nd, Sm, Eu, Dy, Ho, or Er) (Napier et al., 1975). Measurement of the decay time of the D4 level of the terbium(III) ion indicates that transfer takes place from that level to the excited levels of the other rare earths with bimolecular rate constants of 0.5-4.9x lO dm mol s. The fluorescence lifetime for the D4 state of terbium in gaseous Tb(DPM)3 has also been determined. These measurements have been made by Jacobs et al. (1975) as a function of temperature and pressure and the results demonstrate that intermolecular collisional deactivation is not important. Rather, the non-radiative deactivation is an intramolecular process and occurs by means of a transfer to low-lying excited states of the chelate. The fluorescence decay time is 1 s at 200°C which is very much shorter than those observed in 95% ethanol ( 600 /ts) and in the solid state (—500 fis) at room temperature. 

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

Peterson and Bridenbaugh (92) observed a distinct rise and decay in fluorescence from the 5D4 state of terbium in the compound KLa0.9g-Tbo.o2W208 (Fig. 21). Data were collected at room temperature using a stroboscopic technique. Excitation in the form of 5 /usee bursts was applied simultaneously to the 57)3 state and to a few higher levels. The conclusion again was that there was slow internal conversion from the 5Z)3 to the... 

Rieke and Allison (97) studied the fluorescent lifetime of terbium in the chelate terbium trianthranilate (TbAn3). They concluded that the spectra and fluorescent lifetime of the chelate differ markedly from unchelated terbium compounds. Fluorescent-lifetime measurements were made at 25°C, 0°C, and 77°K using a stroboscopic light source with a decay time of 20 /xsec and a comparison was made with TbCl3 4H20. At all... 

The rates of internal conversion from the 5Z)3 to the 5D4 states were also measured. The backup oxide in this case was yttrium. This information was obtained by determining the rise time of the 5Z)4-state green fluorescence as a function of time, when the 5Z>3 state was excited. The rise time of the 5Z)4 state is, of course, the decay time of the 5Z>3 state. It was assumed that the decay of the 5Z)3 was predominantly due to an efficient internal conversion process to the 5D4. Measurements of the decay time of the 5Z)3 state directly were not possible, since the emission from this state is very weak if not, indeed, absent. The result of this study is shown in Fig. 23, where it can be seen that the internal-conversion time remains constant at about 17 fxsec up to a terbium oxide concentration of 1 mole per cent. At higher concentrations, the internal conversion time falls rapidly, until at 10 mole per cent terbium oxide the value is about 1.7 /xsec. This is down by a factor of 10 over samples containing 1 mole per cent or less of terbium oxide. 

Metal complexes like lanthanide chelates (mainly europium or terbium), ruthenium phenanthrolines or bipyridyls, and platinum porphyrins can be used as fluorescent labels for biomolecules. Their long decay times are perfectly suited for a detection by time-resolved imaging, and the labeled target molecules can be used for the determination of intracellular recognition processes or for the screening of DNA and protein arrays. Ratiometric lifetime-based imaging methods in combination with sophisticated data acquisition and evaluation tools can substantially contribute to the development... 

Due to energy backflow problem, the decays of even the stable fluorescent Tb chelates vary rather widely from below 100 ps to as long as 2.6 ms [21, 23, 25]. Fluorescent terbium chelates have also found commercial applications in HTS in Lanthascreen product line of Invitrogen and later on by CisBio with the Lumi4 chelates of Lumiphore company. 

Lanthanide chelates with long decay times can also be used to make efficient tandem probes where both emission wavelength and decay times can be stipulated by selecting the spacer arm and distances between dmior moiety and acceptor. Selvin reported a series of tunable probes composed of fluorescent terbium- and europium-chelates coupled to fluorescein or Cy-5, respectively [32]. 

Window-optimized measurement is accomplished in a dual-label hybridization with dual-label (donor-quencher) probes composed of either europium or terbium donors and fluorescent or nonfluorescent acceptors. In unhybridized free form, flexibility of the probe allows very efficient energy transfer resulting in very short-lived sensitized signal. Upon hybridization, the energy transfer is decreased and decay is longer [43]. Figure 5 shows the assay principle and the respective decays in an assay. 

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