Monitoring of Orientation in Molecular Ensemblesby Polarization Sensitive Nonlinear Microscopy

We present high resolution two-photon excitation microscopystudies combining two-photon fluorescence (TPF) and secondharmonic generation (SHG) in order to probe orientationaldistributions of molecular ensembles at room temperature. Adetailed polarization analysis of TPF and SHG signals is used inorder to unravel the parameters of the molecular orientationalstatistical distribution, using a technique which can be extendedand generalized to a broad variety of molecular arrangements. Apolymer film containing molecules active for TPF and/or SHGemission is studied as a model system. Polarized TPF is shown toprovide information on specific properties pertaining toincoherent emission in molecular media, such as excitationtransfer. SHG, being highly sensitive to a slight departure fromcentrosymmetry such as induced by an external electric field inthe medium, complements TPF. The response of each signal to avariable excitation polarization allows investigation of molecularbehavior in complex environments which affect their orientationsand interactions.