Fluorescence lifetime determinations

Fluorescence Studies. Fluorescence spectra of films on glass plates were obtained with a Perkin-Elmer MPF-3 spectro-fluorimeter. A previously-described phase fluorimeter was utilized for fluorescence lifetime determinations. 

Lifetime studies in the gas phase at low pressures indicated two decay rates 31>. The short-lived component (r = 10 /tsec) has a lifetime an older of magnitude larger than the fluorescence lifetime determined from the absorption spectrum. The longer-lived component has r 200 /nsec. In the model proposed to account for these results, the short-lived component is said to derive from the strong coupling of the singlet level with only a small number n of triplet levels, such that the observed lifetime is given by... 

Measurement of r in the absence of the electron donor will therefore yield k, and in its presence, obs will be obtained, from which k i can be evaluated. However, the proviso discussed earlier concerning product identity still applies, and the wise investigator will be concerned with whether it is electron transfer that is actually causing the quenching process. In the past twenty years, time-resolved measurements for fluorescence lifetime determination have become highly developed and they have been much used in electron-transfer research. 

Figure 2. Schematic diagram of fluorescence lifetime determination apparatus. LI and L2 are lenses M, mirror BST, beam steerer BSP, beam splitter SCH, sample cell holder MONO, monochromator PMT, photomultiplier tube. Other components are described in text.

The use of multifrequency cross correlation phase and modulation phosphorometry, based on a technology usually associated with fluorescence lifetime determination, has also now been applied to the measurement and analysis of triplet state decay times . ... 

Llopis, S. D., Stryjewski, W., and Soper, S. A. Near-infrared time-resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices. Electrophoresis 25, 3810-3819 (2004). 

For all samples of PNVC there is a high energy structureless emission band at 370 nm and, depending on the method of preparation, a more or less significant second structureless emission band at 420 nm (Fig. 8). Fluorescence lifetime determinations show clearly that both emission bands originate from exdmer-like structures and are in complete contrast with the typical highly structured isolated carbazole fluorescence Several interpretations of the origins of the two excimer bands... 

There are, however, problems associated with this method of determining fluorescence lifetimes. First, the phase method is not generally applicable for nonexponential signals and, as we shall see later, there are many cases where the observed fluorescence decay is indeed nonexponential. Second, the method... 

Once the fluorescence quantum yield has been determined, all that is required to calculate the fluorescence rate constant kf is the fluorescence lifetime rf. Direct measurement of this quantity, like the measurement of the fluorescence quantum yield, is difficult, in this case because of the short lifetime of the fluorescent state (shorter than the normal flash from a flash lamp ). There are, however, several methods which have been developed to determine fluorescence lifetimes and these will be the subject of this section. 

This is the best method for determining fluorescence lifetimes. Developed only five or so years ago, this method has achieved wide acceptance/6 A schematic diagram of the instrumentation used in this method is shown in Figure 2.16. 

In a recent evaluation of this phenomenon, the whiteness indices given by eleven individual brighteners on polyester were compared with those of their binary mixtures in various ratios. In many cases the whiteness performance of a mixture was markedly superior to that shown by the individual components [57]. A more specific investigation was confined to a series of benzoxazole FBAs. Their fluorescence spectra and fluorescence lifetimes were determined individually and in mixtures. The relationships between molecular structure and photophysical properties were discussed [58]. 

Fluorescence Lifetimes. Fluorescence lifetimes were determined by the phase shift method, utilizing a previously-described phase fluorimeter. The emission from an argon laser was frequency doubled to provide a 257 nm band for excitation. Fluorescence lifetimes of anisole and polymer 1 in dichloro-methane solution were 2.2 and 1.4 nsec, respectively. Fluorescence lifetimes of polymer films decreased monotonically with increasing DHB concentration from 1.8 (0) to 0.7 nsec (9.2 x 10 3 MDHB). Since fluorescence lifetimes (in contrast to fluorescence intensities) are unaffected by absorption effects of the stabilizer, these results provide direct evidence in support of the intensity measurements for RET from polymer to stabilizer. 

Anisotropy describes the rotational dynamics of reporter molecules or of any sensor segments to which the reporter is rigidly fixed. In the simplest case when both the rotation and the fluorescence decay can be represented by single-exponential functions, the range of variation of anisotropy (r) is determined by variation of the ratio of fluorescence lifetime (xF) and rotational correlation time ([Pg.9]

Thus, when we determine the rate of fluorescence decay, by measuring the fluorescence lifetime, we are measuring the total rate. 

Hanley, Q. S. and Clayton, A. H. A. (2005). AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers. J. Microsc. 218, 62-7. 

Seybold, P. G., Gouterman, M. and Callis, J. (1969). Calorimetric, photometric and lifetime determinations of fluorescence yields of fluorescein dyes. Photochem. Photobiol. 9, 229-242. 

In previous chapters it was shown that FRET can be reliably detected by donor fluorescence lifetime imaging. Here, we will focus on what is perhaps the most intuitive and straightforward way to record FRET imaging of sensitized emission (s.e., that is, the amount of acceptor emission that results from energy transferred by the donor through resonance) by filterFRET. While simple in principle, determinations of s.e. are complicated by overlap of excitation and emission spectra of the donors and acceptors, and by several imperfections of the recording optics, light sources and detectors. 

Tramier, M., Zahid, M., Mevel, J. C., Masse, M. J. and Coppey-Moisan, M. (2006). Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microsc. Res. Tech. 69, 933-9. 

Lifetime [3,9-11] based sensors rely on the determination of decay time of the fluorescence or phosphorescence. Typically, the fluorescence lifetime is 2-20 ps and phosphorescence lifetime is 1 ps to 10 s. Lifetime-based sensors utilize the fact that analytes influence the lifetime of the fluorophore. Thus all dynamic quenchers of luminescence or suitable quenchers can be assayed this way. The relationship between lifetimes in the absence (t0) and presence (t) of a quencher is given by Stern and Volmer ... 

FRET manifests itself through the quenching of donor fluorescence and a reduction of the fluorescence lifetime, accompanied by an increase in acceptor fluorescence emission. The efficiency of the energy-transfer process varies in proportion to the inverse sixth power of the distance separating the donor and acceptor molecules. Consequently, FRET measurements can be utilised as an effective molecular ruler for determining distances between molecules labelled with an appropriate donor and acceptor fluorophore, provided they are within lOnm of each other. 

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