We present a detailed study of two-color two-photon excited fluorescence in indole dissolved in propylene glycol. Femtosecond excitation pulses at effective wavelengths from 268 to 293.33 nm were used to populate the two lowest indole excited states (1)La and (1)Lb and polarized fluorescence was then detected. All seven molecular parameters and the two-photon polarization ratio Ω containing information on two-photon absorption dynamics, molecular lifetime τf, and rotation correlation time τrot have been determined from experiment and analyzed as a function of the excitation wavelength. The analysis of the experimental data has shown that (1)Lb-(1)La inversion occurred under the conditions of our experiment. The two-photon absorption predominantly populated the (1)La state at all excitation wavelengths but in the 287-289 nm area which contained an absorption hump of the (1)Lb state 0-0 origin. The components of the two-photon excitation tensor S were analyzed giving important information on the principal tensor axes and absorption symmetry. The results obtained are in a good agreement with the results reported by other groups. The lifetime τf and the rotation correlation time τrot showed no explicit dependence on the effective excitation wavelength. Their calculated weighted average values were found to be τf = 3.83 ± 0.14 ns and τrot = 0.74 ± 0.06 ns.
The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths, fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function (IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H, are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside the spores, is revealed.
The paper presents experimental and theoretical studies of two-photon excitation dynamics in 2-methyl-5-tert-butyl-p-quaterphenyl (DMQ) dissolved in cyclohexane/paraffin. Experimentally, a two-color two-photon (2C2P) excitation by two femtosecond laser pulses at 800 and 400 nm has been used in combination with the time-resolved detection of polarized molecular fluorescence. The fluorescence decay was found to be two-exponential, resulting in the molecular excited state lifetime of 753 ± 10 ps and the rotational correlation time of 724 ± 45 ps. Control over the excited and fluorescent photons polarization has been used for determination from experiment of seven independent molecular parameters. The experimental data were analyzed on the basis of the recent theoretical approach [Shternin, P. S., Gericke, K.-H., and Vasyutinskii, O. S. Mol. Phys. 2010, 108, 813-825] supported by ab initio computations of the DMQ electronic structure and transition dipole moments. The results obtained imply that the two-photon absorption tensor S is mostly diagonal and that the Szz tensor component onto the molecular long axis gives the major contribution of 93%. However, it was also found that a number of different symmetry two-photon transitions related to the dipole moment components dxdz and dydz are excited in the conditions of our measurements.
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