Two-photon induced fluorescence

Callis P R 1997 Two-photon induced fluorescence Ann. Rev. Rhys. Chem. 48 271-97... 

Figure 8.2e shows the dependence of the fluorescence intensity on the excitation power of the NIR light for the microcrystals measured with a 20x objective. In this plot, both axes are given in logarithmic scales. The slope of the dependence for the perylene crystal is 2.8, indicating that three-photon absorption is responsible for the florescence. On the other hand, slopes for the perylene and anthracene crystals are 3.9 for anthracene and 4.3 for pyrene, respectively. In these cases, four-photon absorption resulted in the formation of emissive excited states in the crystals. These orders of the multiphoton absorption are consistent with the absorption-band edges for each crystal. The four-photon absorption cross section for the anthracene crystal was estimated to be 4.0 x 10 cm s photons by comparing the four-photon induced fluorescence intensity of the crystal with the two-photon induced fluorescence intensity of the reference system (see ref. [3] for more detailed information). 

Patel SA, Richards Cl, Hsiang J-C, Dickson RM (2008) Water-soluble Ag nanoclusters exhibit strong two-photon-induced fluorescence. J Am Chem Soc 130 11602-11603... 

Xu, C., and Webb, W. W. 1997. Nonlinear and two-photon induced fluorescence, Vol. 5. New York Plenum Press. 

The term 2PA spectrum will be used here and in the rest of the chapter to indicate the variation of the two-photon cross section as a function of wavelength, even when the data were obtained by monitoring the intensity of the fluorescence emission induced by two-photon absorption (that is when, strictly speaking, the two-photon induced fluorescence excitation spectrum was obtained). Also, unless otherwise specified, the spectra are reported as a function of the wavelength of the excitation beam and are degenerate 2PA spectra. 

Comprehensive experimental investigations of 2PA processes in fluorene derivatives were performed by Hales et al. [53,56-59] with open aperture Z-scan [26], two-photon induced fluorescence [60] and femtosecond white-light continuum pump-probe methods [61]. For degenerate two-photon excitation, the experimental 2PA spectra of symmetrical and asymmetrical fiuorenes are presented in Figs. 15 and 16. These spectra were obtained with the combination of open aperture Z-scan and two-photon fluorescence methods [57]. For centrosymmetric molecules, two-photon transitions from... 

Fig. 21 Structures of hydrophilic 2PA fluorophores 49 used for cell staining, and confocal microscopy images of live NT2 cells incubated with a two-photon absorbing hydrophilic probe 49 and two-photon induced fluorescence image of fixed NT2 cells (60 x oil) stained with the hydrophilic probe 49...

Fig. 2. Intensity of the two-photon induced fluorescence of HPTS solution at pH 10 (a) and pH 6 (b) are plotted as a function of S. Simulations (continuous lines) based on Eq. 1 are in good agreement with the experimental data (dots).

The P tensor components are obtained from the slope of the scattered light intensity at harmonic frequency versus the square of the fundamental beam intensity, measured for different polarization configurations. The value of the g parameter intervening in Eqs. (9)-( 11) is obtained by calibration with a solvent of known /lvalue. These HRS measurements have to be done very carefully, as other processes such as two-photon-induced fluorescence, Raman, and higher order NLO processes can contribute to the measured HRS signal. Actually, the use of the time-re-solved technique with femtosecond pulses allows for the separation of photons coming from the harmonic generation of the others. 

Fig. 7. Intensity dependence of two-photon induced fluorescence on the pumping light intensity. From [38]...

Because of this wide range of applications, much effort was dedicated to the design and synthesis of new molecules with optimized TPA efficiency in this context, the characteristics of the designed molecules (linear absorption, solubility, substituents...) will depend on the targeted application. The TPA response of moleciUes can be imderstood in the context of its TPA cross-section ajpA. which can be measmed using different techniques, such as nonlinear transmission, two-photon induced fluorescence and the Z-scan method although in a pure TPA process ajpA does not depend on the laser pulse duration, the nonlinear absorption can be more efficient in the nanosecond regime than in the femtosecond one, due to excited state reabsorption phenomena [34]. 

Figure 11.14 Photochemical un-caging using two-photon excitation. Inactive ( caged ) fluorescent probes within the focal volume of the laser (a) are first activated by two-photon-induced photochemical un-caging (b) diffusion of the probes from the focal volume is then monitored by subsequent two-photon-induced fluorescence...

Fig. 6.69 Measurement of short pulses via two-photon induced fluorescence...

Synthesis and nonlinear optical characterization of a two-photon absorbing organic dye, trans-4-(dimethylamino)-4 -[N-ethyl-N-(2-hydroxyethyl)amino]stilbene (DMAHAS), were reported [49]. Linear absorption, single-photon-induced fluorescence, and two-photon-induced fluorescence of DMAHAS were experimentally studied. This dye showed a moderate two-photon absorption cross section of a2 = 0.91 X 10-46 cm s/photon at 532 nm as shown by an open aperture Z-scan technique. DMAHAS also showed strong two-photon-induced blue fluorescence of 432 nm when pumped with 800 nm laser irradiation. 

J. R. T. Lakowicz, Nonlinear and two-photon induced fluorescence, in Topics in Fluorescence Spectroscopy, Plenum Press, New York, 1997, pp. 87-144. 

Lukomska J, Gryczynski I, Malicka J et al (2005) Two-photon induced fluorescence of Cy5-DNA in buffer solution and on silver island films. Biochem Biophys Res Commnn 328 78-84... 

Two-photon induced fluorescence LSCM imaging through the assembled multilayer structure... 

Similar spectral techniques as discussed for macroscopic tumour imaging can be employed for fluorescence microscopy. Confocal and two-photon-induced fluorescence microscopy [10.210], and imaging Fourier transform spectroscopy [10.211] are all valuable techniques for studies at the cellular level. Related to this field is the optical trapping of ceils with focused laser beams optical tweezers), which relies on gradient forces of the same kind as discussed in Sect. 9.8.5. Trapped cells and polymer strings can be manipulated in many ways to enable fundamental studies to be conducted [10.212]. 

J.R. Lakowicz (ed.) Topics in Fluorescence Spectroscopy, Vol. 1 Techniques, VoL 2 Principles, Vol. 3 Biochemical Applications, Vol. Probe Design and Chemical Sensing, Vol. 5 Nonlinear and Two-Photon-Induced Fluorescence (Plenum, New York 1991—1997)... 

Callis, P.R. On the theory of two-photon induced fluorescence anisotropy with application to indoles. J. Chem. Phys. 99, 27-37 (1993)... 

Instead of SHG in nonlinear crystals, often two-photon induced fluorescence (see Sect.8.5) is used to monitor and to visualize the pulse profiles of picosecond pulses [11.25]. The experimental arrangement is shown in Fig. 11.21. The liquid dye solution has no absorption at the fundamental frequency 0) but shows fluorescence at high light intensity due to two-photon absorption. For two identical optical pulses moving into opposite direction through the dye solution the observed fluorescence intensity as a function of the delay time t is again [11.25] given by... 

In the photochemistry of hydrazine induced by an ArF laser, strong two-photon-Induced fluorescence from excited radicals NH(A H) was observed and attributed to secondary absorption by the N2H3 primary photoproduct according to N2H3 + hv-> NH2+NH [22] for critical remarks, see [23, 24]. 

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