ABSTRACT
Polarization-resolved coherent anti-Stokes Raman scattering (CARS) is usually applied to measure the depolarization ratio in solutions or evidence orientation effects in anisotropic media. We present an extensive approach based on multiple-field polarization-resolved CARS, in order to unravel the complexity of vibrational resonances up to the fourth-order symmetry, at the microscopic scale in nonisotropic media. The CARS polarized signals measured under a continuous variation of the incident pump and/or Stokes excitation beams are analyzed using a full tensorial picture both in the nonresonant and resonant regimes. This method evidences the strong influence of vibrational symmetries on polarized CARS, and more specifically the occurrence of Kleinman symmetry deviations at the vicinity of the Raman lines frequencies. This technique, illustrated on a cubic symmetry crystal, is general and can be applied to other medium symmetries.
ABSTRACT
We analyze the influence of the anisotropy of molecular and biological samples on polarization resolved nonlinear microscopy imaging. We show in particular the detrimental influence of birefringence on Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) polarization resolved microscopy imaging, which, if not accounted for, can lead to an erroneous determination of the sample properties and thus to a misinterpretation of the read-out information. We propose a method to measure this birefringence and account for this effect in nonlinear polarization resolved experiments.
Subject(s)
Collagen/chemistry , Imaging, Three-Dimensional/methods , Microscopy, Polarization/methods , Nonlinear Dynamics , Animals , Birefringence , Crystallization , Fluorescence , Photons , Rats , Rats, Sprague-DawleyABSTRACT
We present a global analysis of experimental factors affecting polarization responses in two-photon inverted microscopy. The role of reflection optics and high numerical aperture focusing is investigated in two-photon fluorescence, which can be extended to other nonlinear processes. We show that both effects strongly distort polarization responses and can lead to misleading extraction of molecular order information from polarimetric measurements. We describe a model accounting for these effects and develop a calibration technique for the determination of polarization parameters in the sample plane using two-photon fluorescence polarimetry in liquids.