High-speed polarization-resolved third-harmonic microscopy

Abstract : Polarization-resolved third-harmonic generation (P-THG) is a sensitive probe of material anisotropy and molecular ordering. Despite its promises, this property has little been used in biological tissues due to the lack of measurement schemes compatible with dynamic samples. We report here on the development of a fast P-THG microscope where excitation polarization is switched between line scans using an electro-optic modulator, providing temporal resolution in the 10 ms range for the polarimetric measurement. We demonstrate novel applications enabled by this approach, associated with Fourier-based analysis: probing molecular order in deforming lipid structures undergoing phase transition; revealing crystallinity of flowing particles in the zebrafish embryo’s inner ear; and detecting birefringence in vivo. These results establish that P-THG is ideally suited for probing lipid organization and mineralization in dynamic biological environments.