Lifetimes, spectrally resolved

Instrumentally, spectral FLIM generates a spectrally resolved set of lifetimes by either introducing filters to provide spectral resolution or a spectrograph between the sample and image intensifier. The first such system was created for looking at the long lifetimes of lanthanide dyes [37]. Later, a spectral FLIM system was described for measuring from a two-dimensional (2D) area of a microscope field... 

Hanley, Q. S., Arndt-Jovin, D. J. and Jovin, T. M. (2002). Spectrally resolved fluorescence lifetime imaging microscopy. Appl. Spectrosc. 56,155-66. 

Hanley, Q. S. and Ramkumar, V. (2005). An internal standardization procedure for spectrally resolved fluorescence lifetime imaging. Appl. Spectrosc. 59, 261-6. 

The above calculations, in which the possible participation of a HO(Br)COt intermediate was confirmed, provide a means of reconciling the different rate measurements. For example, with equal A and A" photoexcitation yields, HO(Br)CO+ can be formed as much as half the time. However, this is an upper limit, and it is more likely that HO(Br)COf is formed less than half the time. If these intermediates contribute rotationally cold OH, for example, via reactions (3d) and (3e), the spectrally resolved observation of OH (N = 1) can yield longer lifetimes than the measurements in which all rotational levels are probed simultaneously. This explanation is consistent with all of the experimental observations. However, though it can reconcile the Scherer et al. and Ionov et al. rate measurements, we emphasize that proof is still needed. 

Fig. 5. Energy level diagram for Pd(2-thpy)2 dissolved in n-octane. The Tj state at 18,418 cm is zero-field split on the order of 0.2 cm. The emission decay times refer to the individual triplet suhstates I, II, and III, respectively, at T = 1.3 K. (Compare Fig. 6.) These suhstates are radiatively deactivated as purely electronic transitions, as well as by Franck-Condon (FC) and Herzberg-Teller (HT) vibrational activity, respectively. This leads the different vibrational satellites. (Compare also Sects. 4.2.2 and 4.2.3.) The lifetime of the S, state is determined from the homogeneous linewidth of the spectrally resolved Sq —> S, electronic origin. (Sect. 3.2) The electronic state at 24.7 x 10 cm is not yet assigned...

A two-photon microscope with multispectral FLIM and nondescanned detection is described in [60]. An image of the back aperture of the microscope lens is projected into the input plane of a fibre. The fibre feeds the light into a polychro-mator. The spectrum is detected by a PML-16 multianode detector head, and the time-resolved images of the 16 spectral channels are recorded in an SPC-830 TCSPC module. Spectrally resolved lifetime images obtained by this instrument are shown in Fig. 5.82. 

In combination with advanced TCSPC, the systems can be used to record fluorescence decay curves, dynamic changes of fluorescence decay curves, fluorescence correlation in combination with fluorescence lifetime, and spectrally resolved fluorescence decay profiles. Examples for dynamic lifetime measurements and spectrally resolved lifetime measurements by a multianode PMT with routing are shown under Sects. 5.4.1, page 90, and 5.2, page 84. 

One alternative method is to use differences in fluorescence lifetime as the metric to discriminate between the fluor-labels [138,139]. Several potential benefits of measuring lifetimes for DNA sequencing have been described [140,141]. Lifetimes may be accurately determined in the absence of spectral discrimination thus, discrete lifetimes may be determined for mixtures of fluors coeluting in a single peak. Lifetimes are also independent of intensity even at concentrations where relatively poor SNR may lead to an erroneous fluorescence intensity-based call. In addition, since scattered light has an effective lifetime of zero, scatter may be temporally rejected and is thus less of an issue. However, this method requires a family of fluors in which the lifetimes are resolvable [141-143] and the use of relatively complex electronics [141]. 

Knemeyer, JP, Herten, DP, and Sauer, M, Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy. Analytical Chemistry 75 (2003) 2147-2153. 

Analogous to this development is the combination of spectrally measurements with FLIM (sFLIM, [58,59]), resulting in spectrally resolved lifetimes. In practice, this can be done relatively easy, by inserting a spectrograph or dispersing element between the object and the detector. 

The use of short (fs) laser pulses allows even highly transient ion-radical pairs with lifetimes of t 10 12 s to be detected, and their subsequent (dark) decay to products is temporally monitored through the sequential spectral changes. As such, time-resolved (ps) spectroscopy provides the technique of choice for establishing the viability of the electron-transfer paradigm. This photochemical (ET) mechanism has been demonstrated for a variety of donor-acceptor interactions, as presented in the foregoing section. 

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. 

In the case of NOS 12, different kinetics were observed at 436 nm. In cyclohexane, there was a rapid rise, with a lifetime of 6.6 psec followed by a decay with a 100-psec lifetime. In 1-butanol, there was a rapid rise (lifetime=4.3 psec), a decay (43-psec lifetime), and a second longer decay within a 1.4-nsec lifetime. These findings were confirmed by picosecond time-resolved resonance Raman spectroscopy. In these Raman studies in cyclohexane, a single rate constant was observed, whereas in 1-butanol, three spectral components grew with different time constants. The data were said to be consistent with the photo-formation of two or three isomers trans about the central methine bond however, other transient species could be responsible for the observed kinetics because the absorption envelope obviously shifts and this would affect the resonance Raman bands. 

Other studies, such as infrared and Raman spectra of gaseous benzene, neutron diffraction studies of crystalline benzene, and electron diffraction and microwave spectral studies, are equally incapable, according to critical analysis [87AG(E)782], of resolving unanimously the Dih—Deh structural dilemma of the benzene molecule. Furthermore, no decisive conclusion could be drawn from photoelectron spectra or H—NMR spectrum measurements of benzene molecules in a liquid crystal environment. The latter experiments merely indicate that the average lifetime of a Dih structure (if it appears on the PES) is less than 10 4 sec corresponding to the energy barrier of the Dih- >D6h-+D h interconversion of approximately 12 kcal/mol. 

However, luminescence lifetime, which is a measure of the transition prob-abihty from the emitting level, may be effectively used. It is a characteristic and unique property and it is highly improbable that two different luminescence emissions will have exactly the same decay time. The best way to determine a combination of the spectral and temporal nature of the emission is by using laser-induced time-resolved spectra. The time-resolved technique requires relatively complex and expensive instrumentation, but its scientific... 

To study the excited state one may use transient absorption or time-resolved fluorescence techniques. In both cases, DNA poses many problems. Its steady-state spectra are situated in the near ultraviolet spectral region which is not easily accessible by standard spectroscopic methods. Moreover, DNA and its constituents are characterised by extremely low fluorescence quantum yields (<10 4) which renders fluorescence studies particularly difficult. Based on steady-state measurements, it was estimated that the excited state lifetimes of the monomeric constituents are very short, about a picosecond [1]. Indeed, such an ultrafast deactivation of their excited states may reduce their reactivity something which has been referred to as a "natural protection against photodamage. To what extent the situation is the same for the polymeric DNA molecule is not clear, but longer excited state lifetimes on the nanosecond time scale, possibly of excimer like origin, have been reported [2-4],... 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

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