Multi-photon fluorescence excitation

Bioanalytical Applications of Multi-photon Fluorescence Excitation (MPE)... 

The NIR femtosecond laser microscope realized higher order multi photon excitation for aromatic compounds interferometric autocorrelation detection of the fluorescence from the microcrystals of the aromatic molecules confirmed that their excited states were produced not via stepwise multiphoton absorption but by simultaneous absorption of several photons. The microscope enabled us to obtain three-dimensional multiphoton fluorescence images with higher spatial resolution than that limited by the diffraction theory for one-photon excitation. 

The total fluorescence intensity saturated around a few hundreds of mJ/cm2 which corresponds to the irradiation condition where the new plasma-like emission was observed. Above this value fluorescence intensity decreased, which is accompanied with the recovery of the relative intensity of excimer emissions. This means that a quite efficient deactivation channel of excitation intensity opens in this energy range, and the contribution of Si -Si annihilation is depressed. This suggests that fragmentation reactions to diatomic radicals are not induced by the annihilation process. Multi-photon absorption processes via the Si states and chemical intermediates should be involved, although no direct experimental result has as yet been obtained. 

Valuable findings on the electronic ground and excited states of clusters have been derived from laser-induced multi-photon ionization (MPl) investigations, such as laser-induced fluorescence (LIF) and REMPI. This latter technique is particularly promising since it enables mass selection of cluster species and their spectral and thermochemical characterization. The complex is excited from its electronic ground state from a photon and then ionized by a second photon of equal or different frequency, near threshold to avoid cluster fragmentation. ... 

Lakowicz JR, Gryczynski I, Tolosa L, Dattelbaum JD, Castellano FN, Li L, Rao G. Advances in fluorescence spectroscopy multi-photon excitation, engineered proteins, modulation sensing and microsecond rhenium metal-ligand complexes. Acta Physica Polonica A 1999, 95, 179-196. 

An additional class of nonlinear optical effects is that of multi-photon absorption processes. Using these process, one can create excited states (and, therefore, their associated physical and chemical properties) with a high degree of three-dimensional (3D) spatial confinement, at depth in absorbing media. There are potential applications of multi-photon absorbing materials in 3D fluorescence imaging, photodynamic therapy, nonlinear optical transmission and 3D microfabrication. 

Several articles and reviews on different aspects of multi-photon excitation of biomolecule system are available. For example, Birch [11] consideraticms concentrate mainly on the impact of multi-photon techniques to the time-resolved fluorescence spectroscopy. Lakowicz and Gryczynski [12] have discussed examples of three-photon excited fluorescence. Rehms and Callis studied the two-photon excited fluorescence emission of aromatic amino acids [13]. Kierdasz et al analyzed emission spectra of Tyrosine- and Tryptophan-containing proteins using one-photon (270-3 10 nm) and two-photon (565-6 10 nm) excitation [14]. 

This chapter is devoted to describe the impact of metallic nanosphere to the multi-photon excitation fluorescence of Tryptophan, and little further consideration to multi-photon absorption process will be given, as the reader can find several studies in [11-14]. In section II, the nonlinear light-matter interaction in composite materials is discussed through the mechanism of nonlinear susceptibilities. In section III, experimental results of fluorescence induced by multi-photon absorption in Tryptophan are reported and analyzed. Section IV described the main results of this chapter, which is the effect of metallic nanoparticles on the fluorescent emission of the Tryptophan excited by a multi-photon process. Influence of nanoparticle concentration on the Tryptophan-silver colloids is observed and discussed based coi a nonlinear generalization of the Maxwell Garnett model, introduced in section II. The main conclusion of the chapter is given in secticHi IV. 

Fluorescence emission is just one, but probably the most convenient, of several methods available for observing multi-photon absorption. In a multi-photon excited fluorescence process, the fluorescence intensity Iji does not increase linearly with increasing of the excitation intensity, lac- Instead, Iji and la are related by... 

The excitation of the surface plasmon effect also induces strongly enhanced fluorescence properties of gold nanoparticles due to the enhanconent in the radiative rate of the inter-band electronic transitions relative to that in bulk metals. Metal nanoparticles, especially gold nanorods exhibit enhanced two-photon luminescence (TPL) and multi-photon luminescoice (MPL) [7, 8]. Strongly-enhanced TPL has been observed from individual particles [9, 10] and particle solutions [11] under femtosecond NIR laser excitation. This observation raises the possibility of nonlinear optical imaging in the NIR region, where water and biomolecules have... 

Buehler C, Dreessen J, Mueller K, So PTC, Schilb A, Hassiepen U, Stoeckli KA, Auer M. Multi-photon excitation of intrinsic protein fluorescence and its appUcation to pharmaceutical drug screening. Assay Drug Devel. Technol. 2005 3 155-167. 

High intensity and monochromaticity, resulting in a high spectral intensity, are ideal tools for spectroscopic investigations, especially for fluorescence measurements with low quantum yields, for the study of multi-photon processes and excited states, and for Raman spectroscopy. For example, important biomolecules like nucleic acids have an extremely low fluorescence quantum yield at room temperature. 

The basic idea of this method is to investigate the dependence of the fluorescence intensity on the excitation intensity because this relation determines the order of the non-linearity [8, 38-40]. For example, a quadratic dependence corresponds to a two-photon absorption process and a cubic dependence refers to a three-photon absorption. By means of a tunable excitation light source, one can also map the dispersion of the multi-photon transition, the resulting multi-photon excitation... 

More recently, confocal fluorimetry itself has been impressively extended. In particular, the implementation of multi-photon excitation opened the potential to excite different fluorescent labels by a single laser line [47]. This considerably simplified the optical setup of confocal instruments. For example, Heinze et al. [48] described a setup for two-photon excitation confocal fluorimetry where three molecular species were quantified simultaneously using a single laser. When included in screening systems, these spectroscopic advancements enable the quantification of enzymatic reaction rates on several substrates in parallel or, when applied for peptide or protein ligands, the simultaneous measurement of binding affinities on different target receptors. In this way, biopharmaceuticals can be selected on the basis of their specificity and selectivity. As a consequence, undesired side activities can be controlled very early in the hit identification process. 

The laser combustion diagnostics techniques discussed so far utilized resonant processes, whether it be single- or multi-photon excitation, fluorescence or stimulated emission. We will now consider non-resonant processes of Raman nature. Because of its msensitivity to quenching (the lifetime of the virtual state is lO s), Raman spectroscopy is of considerable interest for quantitative measurements on combustion processes. Further, important flame species such as O2, N2 and H2 that do not exhibit IR transitions (Sect. 4.2.2) can be readily studied with the Raman technique. However, because of the inherent weakness of the Raman scattering process (Sect. 4.3) only non-luminous (non-sooting) flames can be studied. 

According to Eqs. (l)-(3),the generated SHG intensity depends on the square of the incident fight intensity, while the generated THG intensity will depend on the third power of the incident fight intensity. Similar to multi-photon induced fluorescence process, this nonlinear dependence allows localized excitation and is ideal for intrinsic optical sectioning in scanning laser microscopy. Usually the third-order nonlinear susceptibility is much weaker than the... 

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