Molecular orientation sensitive second harmonic microscopy by radially and azimuthally polarized light.

We demonstrate the possibility to switch the z-polarization component of the illumination in the vicinity of the focus of high-NA objective lenses by applying radially and azimuthally polarized incident light. The influence of the field distribution on nonlinear effects was first investigated by the means of simulations. These were performed for high-NA objective lenses commonly used in nonlinear microscopy. Special attention is paid to the influence of the polarization of the incoming field. For linearly, circularly and radially polarized light a considerable polarization component in z-direction is generated by high NA focusing. Azimuthal polarization is an exceptional case: even for strong focusing no z-component arises. Furthermore, the influence of the input polarization on the intensity contributing to the nonlinear signal generation was computed. No distinct difference between comparable input polarization states was found for chosen thresholds of nonlinear signal generation. Differences in signal generation for radially and azimuthally polarized vortex beams were experimentally evaluated in native collagen tissue (porcine cornea). The findings are in good agreement with the theoretical predictions and display the possibility to probe the molecular orientation along the optical axis of samples with known nonlinear properties. The combination of simulations regarding the nonlinear response of materials and experiments with different sample orientations and present or non present z-polarization could help to increase the understanding of nonlinear signal formation in yet unstudied materials.