Abstract

The application range of P-THG microscopy has been so far restricted to studies on molecular order and anisotropy of static specimen removed from their biological environment. Slow polarization commutation limits the investigation of highly dynamic systems because of motion artifacts. Here we have developed a new fast-P-THG microscope enabling efficient in vivo studies in dynamic biological samples. Our P-THG scheme benefits from a built-in EOM that switch polarization states at kilohertz between image lines to provide artefact-free P-THG images with micrometric resolution. Furthermore, we have developed a fast Fourier analysis enabling rapid P-THG processing to quantify lipid order and angular maps. We demonstrated that fast-P-THG is suitable in two major applications. Using first a linear polarization configuration, fast P-THG imaging revealed molecular order changes in MLVs undergoing phase-transition upon heating despite sample distortions. Anisotropy properties of small endogenous microparticles swimming in the otolith cavity embryos were also reported in early zebrafish embryos. A second configuration with linear-circular polarization commutation enabled efficient detection of birefringent media such as anisotropic vesicles in C-elegans gut cells.

© 2019 SPIE/OSA