Excitation two photon

Two-photon fluorescence spectroscopy provides a much better signal-to-noise ratio compared with the one-photon excitation scheme. We have already seen its advantages in the course of measuring the fluorescence of atoms adsorbed near to a metal surface (Section 6.5). In this chapter we shall consider a similar technique based on two-photon evanescent wave excitation of a gas near a surface. Due to the reliability of the signal, this kind of spectroscopy allows one to study the fluorescence lineshapes in detail. 

In gases, the two-photon excitation has an additional advantage when being applied in the geometry of counterpropagating waves. In this case, the overall Doppler broadening is determined by the quantity VtIuii — 012 /c. If 

The intensity of fluorescence induced by the desorbing flux is determined by the quantity A/g/r with Ng the total number of desorbed atoms in the excited state and t their radiative decay lifetime. In a steady-state excitation 

Cunningham, J. M. Morris, J. Fiinfschilling, and D. F. Williams, Chem. Phys. Letters, 

Under normal one-photon excitation conditions, no selective excitation is possible. For two-photon excitation, the moving particle sees each photon of frequency a as a travelling wave of different frequencies w kv, depending on the direction of motion of the particle. If the particle absorbs both photons from a single travelling wave, then the resonance condition is  

Several other papers on the theory of two-photon absorption processes have appeared.459 

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Mikami N and Ito M 1975 Two-photon excitation spectra of naphthalene and naphthalene-dg Chem. Rhys. Lett. 31 472-8... 

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Strickler J FI and Webb W W 1990 Two-photon excitation in laser scanning fluorescence microscopy Proc. SPIE 13948107-18... 

Sanchez E J, Novotny L, Floltom G R and Xie X S 1997 Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation J. Chem. Phys. A 101 7019-23... 

Sonnieitner M, Schutz G J and Schmidt T 1999 Imaging individual molecules by two-photon excitation Chem. Phys. Lett 300 221-6... 

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... 

Mertz J, Xu C and Webb W W 1995 Single-molecule detection by two-photon-excited fluorescence Opt Lett. 20 2532-4... 

Nonlinear optical phenomena, as well as near-field optics, provide us with super resolving capability [20]. The probability of nonlinear optical phenomena is proportional to the number of photons which participate in the phenomenon. For example, the intensity distribution of two-photon excited fluorescence corresponds to the square of the excitation light. Thus, we proposed a combination of the field... 

Sanchez, E. J., Novotny, L. and Xie, X. S. (1999) Near-field fiuorescence microscopy based on two-photon excitation with metal tips. Phys. Rev. Lett., 82, 4014-4017. 

Near-Field Two-Photon Excitation Images of Gold Nanorods 47... 

FigureS.7 Near-field two-photon excitation images of single gold nanorods detected by two-photon induced photoluminescence. Nanorod dimensions (length, diameter) are 540 nm, 20 nm for (a) and 565 nm, 21 nm in (b). Scale bars lOOnm. (Reproduced with permission from The Chemical Society of Japan [11]).

Figure 3.9 Near-field two-photon excitation images of gold nanosphere dimers, (a) Topography. Scale bar 500 nm. (b) and (c) Two-photon excitation images. The excitation wavelength is 780 nm. Incident polarization directions are indicated by arrows. The approximate positions ofthe particles are indicated by circles. (Reproduced with permission from The Japan Society of Applied Physics [12]).

To summarize, we have shown here that enhanced electric-field distribution in metal nanoparticle assemblies can be visualized on the nanoscale by a near-field two-photon excitation imaging method. By combining this method and near-field Raman imaging, we have clearly demonstrated that hot spots in noble metal nanoparticle assemblies make a major contribution to surface enhanced Raman scattering. 

Figure 3.10 (a) Topography of the sample, (b), (c) Near-field excited Raman spectra at dimers 1 and 2, respectively,taken attwodifferentincident polarizations. The peaks marked with are unassigned, (d) Near-field two-photon excitation images of dimers 1 and 2. (e) Near-field Raman excitation images of dimers 1 and 2 obtained for... 

Swift, J. L., Heuff, R. F. and Cramb, D. T. (2006) A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots. Biophys. J., 90, 1396-1410. 

This approach was used by Elliott and co-workers to control the ionization of alkali atoms by one- and two-photon excitation. Wang and Elliott [72] measured the interference between outgoing electrons in different angular momentum states. They showed, for example, that the angular flux of the p2P and the d2D continua of Rb is determined by the phase difference... 

In order to determine the structural factors maximizing 2PA cross section values, we analyze (8) from Sect. 1.2.1. For all cyanine-like molecules, symmetrical and asymmetrical, several distinct 2PA bands can be measured. First, the less intensive 2PA band is always connected with two-photon excitation into the main absorption band. The character of this 2PA band involves at least two dipole moments, /

symmetry forbidden for centro-symmetrical molecules, such as squaraines with C, symmetry due to A/t = 0, and only slightly allowed for polymethine dyes with C2V symmetry (A/t is small and oriented nearly perpendicular to /t01). It is important to note that a change in the permanent dipole moment under two-photon excitation into the linear absorption peak, even for asymmetrical D-a-A molecules, typically does not lead to the appearance of a 2PA band. 2PA bands under the main absorption peak are typically observed only for strongly asymmetrical molecules, for example, Styryl 1 [83], whose S0 —> Si transitions are considerably different from the corresponding transitions in symmetrical dyes and represent much broader, less intense, and blue-shifted bands. Thus, for typical cyanine-like molecules, both symmetrical and asymmetrical, with strong and relatively narrow, S (I > S) transitions, we observe... 

Second, we analyze the nature of the next, strong 2PA bands. The positions of their final states correspond to one-photon symmetry forbidden bands and can be found from excitation anisotropy measurements, as illustrated in Figs. 6,19, and 23. Excitation anisotropy spectra for all cyanine-like molecules typically reveal a large alternation of maximum and minimum features suggesting the positions of the 1PA and 2PA transitions. Two-photon excitation into final states involves two dipole moments, fi0i and /i (i. 

So PT, Dong CY, Masters BR, Berland KM (2000) Two-photon excitation fluorescence microscopy. Annu Rev Biomed Eng 2 399-429... 

Piston DW (1999) Imaging living cells and tissues by two-photon excitation microscopy. Trends Cell Biol 9 66—69... 

Diaspro A, Robello M (2000) Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures. J Photochem Photobiol B 55 1-8... 

Kaiser W, Garrett CGB (1961) Two-photon excitation in CaF2 Eu2+. Phys Rev Lett... 

Xu C, Webb WW (1996) Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. J Opt Soc Am B 13 481—491... 

Kuhn B, Fromherz P, Denk W (2004) High sensitivity of stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge. Biophys J 87(1 ) 631—639... 

Mondal PP, Diaspro A (2007) Reduction of higher-order photobleaching in two-photon excitation microscopy. Phys Rev E 75 6. doi 10.1103/PhysRevE. 1175.061904... 

Patterson GH, Piston DW (2000) Photobleaching in two-photon excitation microscopy. Biophys J 78 2159-2162... 

While fluorescent imaging techniques offer very high sensitivity, there remains the problem of background noise arising from fluorescence from the sample itself (autofluorescence). There are two strategies to overcome this (i) two-photon excitation,32 and (ii) the use of phosphorescent... 

The photophysical properties of magnesium(II) tetra-(i-butyl)phthalocyanine (27) have been studied in solution, in micelles and in liposomes cation radical formation (CBr4 as electron acceptor) has been detected with UV excitation, or by a two-photon excitation using a pulsed laser in the therapeutic window at 670 nm.118 The Mg11 complex of octa(tri-z -propylsilylethy-nyl)tetra[6,7]quinoxalinoporphyrazine (28) has been prepared as a potential PDT sensitizer. The synthesis is shown in Figure 8. Compound (28) has Amax 770 nm (e = 512,000 M-1 cm-1), d>f = 0.46 and d>A = 0.19 (all in THF, under air).119... 

Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 



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