Multiplexed Orthogonal Polarizations Harmonic Imaging for Probing Dynamic Biological Processes

Polarization-resolved nonlinear optical microscopy can provide label-free images of anisotropic biomaterials. Existing techniques are often limited by their complexity, chromaticity, or slowness. In this article, we propose a simple implementation based on the passive splitting of the excitation beam into slightly delayed orthogonally polarized pulse trains. Label-free second- and third-harmonic signals corresponding to each polarization state are separated by time-resolved photon counting, resulting in the acquisition of image pairs that are automatically registered with submicron spatial precision and nanosecond simultaneity. We demonstrate the utility of this small-footprint approach for live studies by presenting structural imaging of tubulin, myelinated axons, and blood vessels in zebrafish larvae, dynamic imaging of flowing microcrystals in the zebrafish larval otolith cavity, and mitotic spindles in dividing embryonic cells.