Fluorescence, time-resolved

Time-Resolved Fluorescence. This instrumental technique may be applied to most immunoassays that require fluorescence intensity measurements, 

A new era of research in fluorescence spectroscopy has emerged with the advent of powerful lasers capable of generating short-lived pulses and with the simultaneous development of sophisticated detection methods. While research groups were previously limited to the study of processes on the microsecond and nanosecond time scale, these developments have expanded the accessible time scale to the pico- and femtosecond. Time-resolved fluorescent measurements are being used, for example, to unravel the dynamics of excited states (excitons) generated in conjugated polymer films (such as stimulated emission) and the processes that 

Time-resolved measurements provide much more information than is available from the steady-state data [7]. For instance, in blends containing two fluorophores, each with a distinct lifetime, it is not always possible to resolve the emission from the two fluorophores using steady-state measurements (if they emit in the same spectral region) and the nature of the processes that occur following photoexcitation. However, the time-resolved data may reveal the individual decay times and the dynamics of generated excited states. 

Two methods of measuring fluorescence lifetime are in widespread use the time-domain (or pulse-fluorometry) and the frequency-domain (or phase-modulation fluorometry) methods. Here, reference will be made only to the first method. 

The instrumentation for time-resolved fluorescence spectroscopy shares some similarities with that used for steady-state measurements, in the sense that an excitation source and a detection systems are used. The nature of these components may, and usually does, vary significantly. Namely, a pulsed light source is used in time-resolved spectroscopy, which is at variance with the continuous light source used in steady-state fluorescence. 

A related experimental method is Time-Correlated Single Photon Counting (TCSPC), which generates a histogram representing the fluorescence intensity over time. This is an efficient method because it counts photons and records their arrival time, which directly represents the fluorescence decay. 

However, since steady-state fluorescence spectra result from the sum of the contribution of the individual species present in the system, the identification of individual species or chemical events (structural alteration, formation or disappearance of molecular species due to chemical reactions) occurring within complex systems may not be straightforward. This limitation may be overcome by using time-resolved fluorescence techniques [7]. 

Time-resolved fluorescence is a time-dependent technique allowing for the determination of events occurring during the excited state lifetime of the fluorophore, which may vary from a few picoseconds to nanoseconds. 

The lifetime (or decay time) of the fluorophores can be obtained using two different time-resolved methods pulse fluorometry (generally, using the single photon timing method) or phase modulation fluorometry. Pulse fluorometry is by far the most popular method therefore the whole discussion developed in this chapter is based on the principles of this method. 

In pulse fluorometry, the sample is excited by a pulsed light beam (5 pulse), provided by a mode-locked laser or by a flash lamp. The fluorescence response to 

The fluorescence decay curve accounts for the contribution of the individual decay times of all fluorescing residues (fluorophores in different environments) decaying to the ground state within the selected measuring time window. Therefore, through this technique it is possible to access to the individual contribution of each fluorescence species, i, in solution, to the overall fluorescence spectra by the signature of their decay times, t . 

A molecule can also be excited by absorbing two photons simultaneously [189]. The sum of the energy of the photons must be larger than the energy gap between SI and SO. Because two photons are required to excite one molecule, the excitation efficiency increases with the square of the photon flux. Efficient two-photon excitation requires a high photon flux, which is achieved in practice only by a pulsed laser and by focusing into a diffraction-limited spot. Due to the nonlinearity of two-photon absorption, the excitation is almost entirely confined to the central part of the diffraction pattern. 

Higher excited states, S2, S3, do exist, but they decay at an extremely rapid rate into the S1 state. Moreover, the electronic states of the molecules are broadened by vibration. Therefore, a molecule can be excited by a photon of almost any energy higher than the gap between SO and SI. 

Without interaction with its environment, the moleeule ean return from the SI state by emitting a photon or by internal conversion of the absorbed energy into heat. The probability that one of these effects will occur is independent of the time after the excitation. The fluoreseenee decay function, measured at a large number of similar molecules, is therefore single-exponential. 

The excited-state lifetime of the molecule in absence of any radiationless deeay processes is the natural fluorescence lifetime , r . The natural lifetime is a constant for a given molecule and given refraction index of the solvent. Because the absorbed energy can also be dissipated by internal conversion, the effective fluorescence lifetime, is shorter than the natural lifetime, The fluorescence quantum efficiency , i.e. the ratio of the number of emitted photons to absorbed photons, reflects the ratio of the radiative decay rate to the total decay rate. Most dyes of high quantum efficiency, such as laser dyes and fluorescence markers for biological samples, have natural fluorescence decay times of the order of 1 to 10 ns. There are a few exceptions, such as pyrene or coronene, with lifetimes of 400 ns and 200 ns, and rare-earth chelates with lifetimes in the ps range. 

There are a number of additional pathways the molecule can use to return to the ground state. The most relevant ones in practice are intersystem crossing and dynamic (or collisional) quenching. 

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Time-resolved fluorescence is perhaps the most direct experunent in the ultrafast spectroscopist s palette. Because only one laser pulse interacts with the sample, the mediod is essentially free of the problems with field-matter time orderings that arise in all of the subsequently discussed multipulse methods. The signal... 

Cross A J and Fleming G R 1984 Analysis of time-resolved fluorescence anisotropy decays Blophys. J. 46 45-56... 

Matro A and Cina J A 1995 Theoretical study of time-resolved fluorescence anisotropy from coupled chromophore pairs J. Phys. Chem. 99 2568-82... 

Loring R F, Van Y J and Mukamel S 1987 Time-resolved fluorescence and hole-burning line shapes of solvated molecules longitudinal dielectric relaxation and vibrational dynamics J. Chem. Phys. 87 5840-57... 

Murakami H, Kinoshita S, Hirata Y, Okada T and Mataga N 1992 Transient hole-burning and time-resolved fluorescence spectra of dye molecules in solution evidence for ground-state relaxation and hole-filling effect J. Chem. Phys. 97 7881-8... 

Brand L, Eggeling C, Zander C, Drexhage K FI and Seidel CAM 1997 Single-molecule identification of coumarin-120 by time-resolved fluorescence detection comparison of one- and two-photon excitation in solution J. Chem. Phys. A 101 4313-21... 

In time-resolved fluorescence, rare earths are frequently used as fluorescent labels. The fluorophores have large Stokes shifts, ie, shifts of the emitted light to a higher wavelength relative to the absorption wavelength, and comparatively long decay times, approximately 0.5 ms. This simplifies the optical... 

Perozzo, M. A., Ward, K. B., Thompson, R. B., and Ward, W. W. (1988). X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals. J. Biol. Chem. 263 7713-7716. 

Ghiggino, K. P., Roberts, A. J. and Phillips, D. Time-Resolved Fluorescence Techniques in Polymer and Biopolymer Studies. Vol. 40, pp. 69— 167. 

Van Paassen [57] describes the CMC of some polyether carboxylates with different fatty chains and EO degrees (Fig. 2). In an extensive study, Binana-Limbele et al. [59] investigated the micellar properties of the alkylpolyether carboxylates of the general formula CnH + OCF CH OCI COONa with n = 8, x = 5, and n = 12 and x = 5,1, and 9, by means of electrical conductivity (CMC, apparent micellar ionization degree) and time-resolved fluorescence probing (micelle aggregation number A7) as a function of temperature and surfactant concentration (Table 1). 

Moya, I. NATO ASI Ser., SerA Time-Resolved Fluorescence Spectroscopy in... 

Brand, L. Laws, W.R. NATO ASI Ser., Ser.A Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology Plenum Press New York, 1983 pp 319-40. 

Time Resolved Fluorescence Depolarization. In Equation 3, it is assumed that the polarization decays to zero as a single exponential function, which is equivalent to assuming that the molecular shape is spherical with isotropic rotational motion. Multiexponential decays arise from anisotropic rotational motion, which might indicate a nonspherical molecule, a molecule rotating in a nonuniform environment, a fluorophore bound to tbe molecule in a manner that binders its motion, or a mixture of fluorophores with different rotational rates. 

E. Analysis of Interfacial Complex by a Time-Resolved Fluorescence Spectroscopy... 

Investigation of water motion in AOT reverse micelles determining the solvent correlation function, C i), was first reported by Sarkar et al. [29]. They obtained time-resolved fluorescence measurements of C480 in an AOT reverse micellar solution with time resolution of > 50 ps and observed solvent relaxation rates with time constants ranging from 1.7 to 12 ns. They also attributed these dynamical changes to relaxation processes of water molecules in various environments of the water pool. In a similar study investigating the deuterium isotope effect on solvent motion in AOT reverse micelles. Das et al. [37] reported that the solvation dynamics of D2O is 1.5 times slower than H2O motion. 

FIG. 4 Time-resolved fluorescence Stokes shift of coumarin 343 in Aerosol OT reverse micelles, (a) normalized time-correlation functions, C i) = v(t) — v(oo)/v(0) — v(oo), and (b) unnormalized time-correlation functions, S i) = v i) — v(oo), showing the magnitude of the overall Stokes shift in addition to the dynamic response, wq = 1.1 ( ), 5 ( ), 7.5 ( ), 15 ( ), and 40 (O) and for bulk aqueous Na solution (A)- Points are data and lines that are multiexponential fits to the data. (Reprinted from Ref 38 with permission from the American Chemical Society.)... 

Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio International) is an assay based on the proximity of a lanthanide cryptate donor and a fluorescent acceptor molecule whose excitation wavelength overlaps that of the cryptate s emission. The utility of this technique is based on the time resolved fluorescence properties of lanthanides. Lanthanides are unique in the increased lifetime of their fluorescence decay relative to other atoms, so a delay in collection of the emission intensity removes the background from other fluorescent molecules. An example of the HTRF assay is a generic protein-protein interaction assay shown in Fig. 2. 

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

Mullineaux, C.W., Pascal, A.A., Horton, P. and Holzwarth, A.R. 1992. Excitation energy quenching in aggregates of the LHCII chlorophyll-protein complex A time-resolved fluorescence study. Biochim. Biophys. Acta 1141 23-28. 

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