The inherent advantages of nonlinear excitation make multiphoton fluorescence microscopy (MPFM) awell-suited imaging technique for extracting valuable information from turbid and thick biological samples. These advantages include high three-dimensional spatial resolution, large penetration depth, minimum out-of-focus cellular photodamage, and high signal-to-noise contrast. We have investigated the nonlinear spectroscopy of biologically important molecules such as NADH, flavins, and intrinsically fluorescent proteins. Fundamental understanding of the molecular spectroscopy and dynamics of these biomolecules is essential for advancing their applications in biological research. MPFM has been utilized for monitoring a large spectrum of biological processes including metabolic activity and exocytosis. We will discuss two-photon (2P) redox fluorescence microscopy of NADH, which gives a quantitative measure of the respiratory chain activity, thus allowing functional imaging of energy metabolism in neurons and native brain tissue. Finally, a rational design strategy, based on donor-acceptor-donor configuration, will be elucidated for fluorescent probes with large 2P-excitation cross-section. These dyes are water-soluble, yet possess a high affinity to organic phases with site-specific labeling and Ca⁺² sensitivity (K_d ~ 350 nM). A brief account on the biological application of nanocrystals and second harmonic imaging will be reviewed.

Initial relaxation kinetics in a series of carotenes has been investigated using femtosecond absorption and Raman spectroscopy. The internal conversion from the photoexcited 1B_u⁺ state to the 2A_g^- state is faster in the carotenes with middle polyene lengths. The vibrational relaxation of the 2A_g^- state has fast and slow components. The time constant of the slow component is longer than several picoseconds. The 2A_g^- state in the short carotenes remains in the vibrational excited levels because of the slow vibrational relaxation. These properties can be explained in terms of the 1B_u⁺ state which assists the internal conversion to the 2A_g^- state and the fast vibrational relaxation of the 2A_g^- state. The vibrational features of the excited states in carotenoids is very important in energy transfer mechanism of acterial photosynthesis.

Novel microscopies based on nonlinear optical (NLO) phenomena are attracting increasing interest in the biology community owing to their potentialities in the area of real-time, non-damaging imaging of biological systems. In particular, second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) are NLO phenomena that scale with excitation intensity squared, and thus give rise to an intrinsic 3-dimensional resolution when used in microscopic imaging. In this perspective, we have implemented a molecular engineering approach toward NLO-probes specifically designed for SHG and/or TPEF imaging of cellular membranes. We have designed nanoscale rod-like fluorophores showing very large TPEF cross-sections in the visible red, outperforming standard fluorophores such as fluorescein by up to two orders of magnitude. Bolaamphiphilic derivatives combining high TPEF cross-sections and affinity for cellular membranes were prepared. Their incorporation into model or cell membranes can be monitored by TPEF microscopy. Amphiphilic push-pull chromophores showing both high TPA and SHG cross-sections in the near-IR region were designed as NLO-probes for imaging of biological membranes by simultaneous SHG and TPEF microscopy. These NLO-phores offer intriguing potentialities for imaging of fundamental biological processes such as adhesion, fusion or for reporting of membrane electrical potentials.

Incoherent second-order nonlinear scattering, or hyper-Rayleigh scattering (HRS) has been evaluated as a new experimental tool for symmetry fluctuation spectroscopy. As such, it is the second-order nonlinear analogue of the linear dynamic light scattering (DLS) of quasi-elastic light scattering (QELS) technique for density fluctuation spectroscopy. High peak power is needed in a short pulse for the second-order nonlinear optical effect to be observed. The periodical structure of the impinging light itself does not impede the measurement for intensity correlation times longer than the pulse-to-pulse period. However, the inherently large spectral bandwidth of femtosecond pulses considerably reduces the amplitude of the autocorrelation function. Reducing the coherence volume to increase this amplitude results in a lower count rate. The low efficiency of the second-order nonlinear light scattering, possible relaxation oscillation in solid-state femtosecond lasers and its quadratic amplification in second-order nonlinear scattering have pronounced influence on the autocorrelation function. Finally, typical relaxation times expected for the dynamics associated with large fluctuations in second-order nonlinearity put a severe limit on the applicability of quasi-elastic nonlinear light scattering for the study of chemical reaction dynamics.

The technique of stimulated emission depletion of fluorescence (STED) from a two photon excited molecular population is demonstrated in the S1 excited state of fluorescein in ethylene glycol and methanol. Two photon excitation (pump) is achieved using the partial output of a regeneratively amplified Ti:Sapphire laser in conjunction with an optical parametric amplifier whose tuneable output provides a synchronous depletion (dump) pulse. Time resolved fluorescence intensity and anisotropy measurements of the fluorescein emission are made using picosecond time-correlated single photon counting. Pump-dump time delayed fluorescence intensity measurements are used to characterise the response of the system and to provide additional data on saturation dynamics of the dump transition. Two photon STED is modelled using both approximate analytical techniques in the weak dump limit and by numerical solutions to the appropriate rate equations. The latter are used to fit experimental data from which it is possible to determine the cross-section for the stimulated transition and lifetime of the upper vibrational levels of the ground state.

Matrix formulation of TDHF theory of HP is given, (2i+1)-rule for dynamic HP and compatibility of TDHF with finite field approach are proven. The definition of tensor invariants of HP is given for the general case. The above results are illustrated by calculations of HP in organic molecules with expressed nonlinear optical properties. A method of inclusion of intermolecular Madelung potential into HP calculation in crystals and crystal layers is described and illustrated by estimation of its influence on HP of some Langmuir-Blodgett films. Similar technique is developed for calculation of Lorentz factors in crystals and crystal layers, which have been used for transformation of molecular HP into higher susceptibilities of crystals and films.

The propensity of conventional optical beams to convey angular momentum is very well known. As a spin-1 elementary particle any photon can assume a polarisation state with a well defined 'spin' angular momentum of plus or minus 1 in the direction of propagation, corresponding to a circular polarisation of either left or right helicity. The mechanical effects of photonic angular momentum are manifest in a variety of phenomena operating at both the atomic and macroscopic scale. Photon angular momentum also exercises a key role in atomic spectroscopy and a host of other fundamental optical phenomena. The aim of this work is to study the interaction between matter and Laguerre-Gaussian beams, and others of related structure in which a helical wavefront confers an endowment with 'orbital' angular momentum. Although the principles and methods of production of these twisted beams are already quite well understood, the detailed study of the interactions is a novel subject. We explore changes in selection rules transfer of linear and angular momentum in the context of nonlinear processes, especially harmonic and sum-frequency generation.

Impulsive stimulated scattering (ISS) has been used to obtain the orientation dependence of the velocity of the surface acoustic waves (SAW) on single crystal metal and semiconductor surfaces. The ISS data is presented for mechanically polished (001) plane of nickel and germanium, and (111) of aluminum at ambient pressure. The orientationally dependent ISS results for Ni (100) are compared with the results of Brillouin scattering measurement as well as calculation results. Finally, the results of high pressure ISS experiments are presented for the SAW on polycrystalline aluminum.

We present a newly developed microscope for sum frequency generation (SFG) imaging of opaque and reflecting interfaces. The sample is viewed at an angle of 60° with respect to the surface normal in order to increase the collected SFG intensity. Our setup is designed to keep the whole field of view (FOV) in focus and to compensate for the distortion usually related to oblique imaging by means of a blazed grating. The separation of the SFG intensity and the reflected visible beam is accomplished by a suitable combination of spectral filters. The sum frequency microscope (SFM) is capable of in-situ chemically selective imaging by tuning the IR-beam to vibrational transitions of the respective molecules. The SFM is applied to imaging of structured self-assembled monolayers (SAM) of thiol molecules on a gold surface.

All-optical poling permits the polar orientation of molecules. FOr an efficient poling of thin films, relative phases, amplitudes and polarizations of the two interfering beams must be controlled. We present an original stable one-arm interfermeter which is specific to the recording of two-color interferences. It relies on teh index dispersion of optical glasses. This particular interference technique permits true real-time non-perturbative monitoring of the polar orientation process and a fast all optical poling of thin film materials, without need for phase control.

Water in oil microemulsions are systems of spherical droplets of water coated by a monolayer of surfactant molecules, immersed in oil. Initially we have studied the optical nonlinear behavior of water in oil microemulsion by the Self-Phase-Modulation of a gaussian laser beam by an optically thin film. The material is WAD (water/AOT/decane, where AOT denotes sodium-bis-di-ethyl-sulfosuccinate) far from critical points and near the percolative transition from electrically insulating to electrically conducting. We have observed optical nonlinearity in the L2 area of the phase diagram, near the percolation line and far from the one-phase two-phase boundary line. In this point, the material turbidity is very low. Strong optical nonlinearity has been reported. Nonlinear optical effects in a Water/AOT/Decane (WAD) microemulsion have been experimentally studied also in a pump probe configuration. We detect the variation of the on axis optical intensity of the probe beam as generated by the concentration profile induced in an optically thin film of microemulsion by the pump beam. Results seem to suggest the hypothesis of a chain like shape of the clusters.

The influence of the environment on vibrational dynamics, and especially on the vibrational dephasing time, is investigated for a simple system : DCl in a van der Waals solid (N₂ lattice). Photon echo experiments are performed using the infrared free electron laser CLIO. In solid nitrogen, decay times of the third order polarization are in the picosecond range. In the case of the isolated DCl monomers in van der Waals interaction with the host, the observed signals are interpreted in term of a rapid spectral diffusion. The two different stretching modes of DCl dimers, perturbed in a different way by the weak hydrogen bond, exhibit different coherence times, reflecting the influence of the interaction strength between the molecules.

There are less than a handful of papers in the literature on the phase measurement of surface Second Harmonic Generation (SHG) from liquid interfaces, in sharp contrast to tens of the works on such measurement of films on solid substrate surfaces. Even though the SHG phase measurement is very important for obtaining structural and spectroscopic information, such as molecular orientation, at the liquid interfaces, experimentally there are many difficulties due to the intrinsically weak SH signal of liquid interfaces. Using a femtosecond pulsed laser and photon counting system, we have demonstrate the ability to measure the SH phase for the non-resonant neat air/water interface, which is clearly among the systems with the smallest surface SH susceptibility, and is well below the detection limit of experimental setups in most laboratories. After clarifying some confusions about the reference standards for absolute SH phase measurement using the α-quartz crystal, we used air/water interface as an intrinsic SH phase reference standard to measure the SH phases of some air/pure-liquid interfaces, and adsorbed molecular layers at the air/water interface. These results demonstrate the effectiveness of this very sensitive technique in obtaining molecular structural and spectroscopic information of liquid interfaces.

Incoherent second-harmonic (SH) and third-harmonic (TH) generation, or hyper-Rayleigh scattering (HRS) on SH and TH, is proposed as a probe of nonlinear-optical properties of silver island films (SIF). HRS on SH and TH indicatrices and linear (Rayleigh) scattering (RS) indicatrices are measured. By the atomic force microscopy (AFM) we apply to study the surface topographic inhomogeneity. The density-density correlation function which describes the correlation of the spatial nonlinear susceptibilities fluctuations is derived from the AFM images of SIF. The interpretation of the experimental results is based on the correlation between optical ) properties and structural features of SIF.

For liquid ethylacetate the frequency maximums for parallel (I_|| (v)) and perpendicular (I_&highmod;(v)) polarized components of C=O vibrations band in Raman spectra are differed on 5.3 cm^-1. At dilution ethylacetate in CCl₄ and heptane or heating in this difference is decreased by displacement of I_|| (v) maximum to the I_&highmod;(v) maximum. In polar solvent, nitrometane, the picture is different - the frequency maxima difference is decreased though the displacement of I_&highmod;(v) band maximum to the I_|| (v)one. The results were explained by the complexity of C=O vibration bands, and existence within the band of two lines with the different depolarization ratio. The complexity of the band is the result existence in liquid ethylacetate the monomer molecules and molecular aggregations.

Quasiempirical calculations of stable molecular aggregations for dimethylsulfoxide were carried out by MINDO/3 method. The structure of the aggregates and energy of its formation for cases of two, three and four molecules were determined. It was shown that except monomer molecules are possible aggregations from two or three molecules. It was established that the aggregations form by charge interaction of polar molecules. Apparently in liquid state of substance the tendency of aggregation formation remains. The presence of three imposed bands in Raman spectra of S=O vibrations for dimethylsulfoxide was connected, on our opinion, with this. The difference of depolarization ratio for imposed bands is connected with different distribution of charge on S=O bond for different aggregations.