Open-source Micro-Manager Plugin for Live-view Imaging of Fluorescent Dipoles.

Abstract

Fluorescence polarization microscopy (FPM) can image the position and dipole orientation of fluorophores. Despite the achievements of super-resolution fluorescence polarization microscopy, their reliance on post-acquisition hinders real-time observation. Polarized structured illumination microscopy (pSIM) offers super-resolution imaging of fluorescent dipoles with fast imaging speed and is well-suited for live-cell applications. We developed an open-source implementation for real-time reconstruction of polarization images and display of the fluorescent dipoles. Additionally, we extended the method to achieve 3D orientation mapping (3DOM), broadening its utility for complex biological studies.Furthermore, we have presented a thorough introduction to extending an existing SIM microscope on polarization imaging and provided a detailed configuration guide of Micro-Manager 2.0 to control the microscope, enabling real-time preview of polarized imaging. Additionally, we have provided the MATLAB code for full reconstructionencompassing both pSIM and 3DOM. This comprehensive guide aims to assist beginners in quickly mastering and easily getting started with the operations.