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

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Fluorescence polarization microscopy (FPM) images fluorophore position and dipole orientation.
• Existing super-resolution FPM methods require post-acquisition processing, limiting real-time live-cell imaging.
• Polarized structured illumination microscopy (pSIM) offers fast, super-resolution imaging suitable for live-cell applications.

Purpose of the Study:

• To develop an open-source implementation for real-time reconstruction and display of polarization images.
• To extend pSIM for 3D orientation mapping (3DOM) for complex biological studies.
• To provide a comprehensive guide for setting up and operating pSIM and 3DOM.

Main Methods:

• Developed an open-source software for real-time pSIM image reconstruction and dipole display.
• Extended pSIM to enable 3D orientation mapping (3DOM).
• Provided a detailed configuration guide for Micro-Manager 2.0 and MATLAB code for reconstruction.

Main Results:

• Achieved real-time reconstruction and display of fluorescent dipole orientation using pSIM.
• Successfully implemented 3D orientation mapping (3DOM) capability.
• Demonstrated a practical setup for extending existing SIM microscopes for polarization imaging.

Conclusions:

• The developed open-source pSIM and 3DOM methods enable fast, real-time super-resolution imaging of fluorescent dipoles.
• The comprehensive guide and provided code facilitate adoption by researchers, including beginners.
• This work broadens the utility of pSIM for advanced live-cell biological studies.