Second-harmonic-generation microscopy using excitation beam with controlled polarization pattern to determine three-dimensional molecular orientation

We have developed a second-harmonic-generation (SHG) microscope using an excitation beam with a controlled polarization pattern in order to detect three-dimensional molecular orientation. The electric field at the focus is controlled three-dimensionally by modifying the polarization distribution with a parallel-aligned nematic-liquid-crystal spatial-light-modulator without any mechanical moving parts. We demonstrated that the SHG signal from an Achilles tendon, sliced so that collagen fibers were aligned parallel to the optical axis, excited by a radially polarized beam was higher than those excited by linearly polarized beams. The possibility of determinating three-dimensional molecular orientation was thus shown.