Time resolved fluorescence composites

To date, there have been only a handful of time-resolved studies in dense fluid media (33,34,69-72). Of these, the bulk have focused on understanding a particular chemical reaction by adjusting the solvent environment (69-71). Only over the past two years have there been experiments directed toward studying the peculiar effects of supercritical fluids on these solvation processes (33,34,72). The initial work (33,34) showed that 1) time-resolved fluorescence can be used to improve our understanding of solvation in supercritical fluids and 2) the local solvent composition, about a solute molecule, could change significantly on a subnanosecond time scale. 

We have utilized the static and dynamic fluorescence characteristics of an environmentally-sensitive solute molecule, PRODAN, to investigate the local solvent composition in binary supercritical fluids. In the two solvent systems studied (C02/1.57 mol% CH3OH and C02/1.44 mol% CH3CN), specific cosolvent-solute interactions are clearly evident. Time-resolved fluorescence emission spectra indicate that the cosolvent-solute interactions become more pronounced with time after excitation. Hence, the local composition of cosolvent around the excited-state solute becomes greater than that surrounding the ground-state solute. That is, the photon-induced increase in excited-state dipole moment drives picosecond cosolvent augmentation about PRODAN. 

Aladan substitution of internal core amino-acid residues provides an approach to characterise the physical characteristics of protein cores. Steady-state fluorescence alone can provide initial insight to the immediate environment of Aladan in the protein core. However, time-resolved fluorescence spectroscopy can be used to understand variations in protein core composition and structure as a function of time through the characterisation of Aladan fluorescence intensity and /max changes that are caused by small fluctuations in the relative permittivity, e, of the protein interior with time (fs-ps timescale). Such spectroscopy is possible since fluorescence lifetimes, Tr, are typically in the ns range (see Section 4.5). Also, time-resolved fluorescence spectroscopy can be performed with non-covalently linked extrinsic fluorophores such as ethidium bromide (EtBr). This fluorophore intercalates between the bases of DNA or RNA double helix and in so doing acquires a substantial increase in (j) and hence fluorescence intensity at /max (595 nm). Should there be a disruption or collapse in double-helical structure, then intercalation fails and fluorescent intensity drops... 

Winnik et al. [53] used time-resolved fluorescence spectroscopy (direct non-radi-ative energy transfer experiments) to determine the interface thickness in films of symmetric poly(styrene-fc-methyl methacrylate) (PS-PMMA) block copolymers labeled at their junctions with either a 9-phenanthryl or a 2-anthryl group. The corrected donor fluorescence decay profiles were fitted to simulated fluorescence decay curves in which the interface thickness 8 was the only adjustable parameter. The optimum value of the interface thickness obtained was 6 = 4.8 run. In similar studies [54—57], the same authors determined the interface thickness value 6 = 1.6 nm in mixtures of two symmetrical poly(isoprene-b-methyl methacrylate) (PI-PMMA) block copolymers of similar molar mass and composition [54] the interface thickness value 8 = 1.1 nm for the lamellar structures formed in films of symmetric PI-PMMA diblock copolymers bearing dyes at the junctions [55] a cylindrical interface thickness value of d slightly smaller than 1.0 nm in films consisting of mixtures of donor- and acceptor-labeled PI-PMMA (29vol% PI) that form a hexagonal phase in the bulk state [56] and the interface thickness 8 = 5 run on the diblock copolymer poly(styrene-l>-butyl methacrylate)(PS-h-PBMA) [57]. 

Time resolved fluorescence measurements have been used for decades because they are such a powerful tool to investigate fluorophore-metal composites. Due to insufficient time resolution, mostly long lived luminescence like that from triplet states has been investigated. When fluorophOTes are attached to metal nanostructures, fluorescence decay times are in the sub nanosecond time range. To measure those dect times accurately, techniques such as time correlated single photon counting, frequency domain fluorescence measurements, streak camera measuremets, and femtosecond pump SHG-probe have been used. 

Figure 8 Time resolved fluorescence signal from a 0.37 /tM Lissamine solution (upper curve) and a mixed solution of 0.37 fiM Lissamine and of0.2S nM gold nanoparticles of r = IS nm radius. The ultrafast spike at t K 0 stems from the gold particles, while the long lived component is from unbound Lissamine molecules. The intermediate decay signal is from Lissamine - nanoparticle composites.

Figure 12 Time resolved fluorescence decay of Rhodamine - gold nanoparticle composite systems of varying spacer length.

Kim and Johnston (27), and Yonker and Smith (22) have used solute solvatochroism to determine the composition of the local solvent environment in binary supercritical fluids. In our laboratory we investigate solute-cosolvent interactions by using a fluorescent solute molecule (a probe) whose emission characteristics are sensitive to its local solvent environment. In this way, it is possible to monitor changes in the local solvent composition using the probe fluorescence. Moreover, by using picosecond time-resolved techniques, one can determine the kinetics of fluid compositional fluctuation in the cybotactic region. 

Absorption spectroscopy and laser induced fluorescence (LIF), give access to the concentration of molecules, atoms, and ions in the ground state. LIF is enable to achieve highly spatial and time resolved analyses. This technique is thus particularly suitable to investigate composition changes in the plasma, and obtain spatial or time concentration profiles. Published results in fluorine plasmas using absorption [25-27] and LIF [28-32] mainly concern temperature measurements [25] or the quantification of CFV radicals [26-31] in fluorocarbon-based plasmas and SOx in SF6—02 discharges [32], Recently LIF has been used to measure plasma-surface interaction products [33]. 

Over the past decade, Raman spectroscopy has continued to develop as a prime candidate for the next generation of in situ planetary instruments, as it provides definitive stmctural and compositional information of minerals in their natural geological context. A time resolved Raman spectrometer have been developed that uses a streak camera and pulsed miniature microchip laser to provide picosecond time resolution (Blacksberg et al. 2010). The ability to observe the complete time evolution of Raman and fluorescence spectra in minerals makes this technique ideal for exploration of diverse planetary environments, some of which are expected to contain strong, if not overwhelming, fluorescence signatures. In particular, it was found that conventional Raman spectra from fine grained clays. 

The fluorescence spectrum of the composite system, i.e. a solution of 0.029 nM unpassivated gold nanoparticles (r = 30 nm) and 0.18 fiM Lissamine dye molecules, is shown by the dotted curve in figure 6. In fact, it is hardly distinguishable from the abscissa. Compared to the fluorescence of the solution with passivated particles, only -5 % of fluorescence intensity is left over. From the following discussion it will become clear that most of this residual fluorescence is due to fluorescence fi-om unbound Lissamine molecules inevitably present due to the thermodynamic equilibrium between bound and unbound molecules. Only time resolved measurements are able to distinguish between fluorescence of bound and unbound molecules. Below it will become clear that the nanoparticles quench the fluorescence of the bound molecules by more than 99 %. 

At the present stage of our understanding of fluorescent-decay processes, the ideas are often of equal or greater importance than the exact nature of the chemical species studied. It did not seem appropriate to attempt to resolve the problem of chemical composition at this time, and essentially the name given to the compounds by the author is used. 

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