4polar3D single molecule imaging of 3D orientation in dense actin networks using ratiometric polarization splitting

Single Molecule Orientation and Localization Microscopy (SMOLM) aims at simultaneously measuring the position and orientation of single molecules, generating orientation-encoded super-resolved images by estimating both their 3D mean orientation and the extent of their angular fluctuations (wobble). Most existing SMOLM approaches rely on the engineering of single molecules' point spread functions, which requires complex optical setups and long computational times that can be an obstacle in dense cellular environments with high detection density and challenging imaging conditions. In this work, we propose a simpler and effective method named 4polar3D, based on the estimation of single molecule intensities projected onto four polarized channels with controlled numerical apertures. This strategy enables 3D orientation measurements of single molecules within a 0-180° azimuthal range in addition to their angular range of fluctuations and their 2D localization, using a setup requiring minimal alignment complexity. It is moreover based on pure intensity-estimation, making data processing considerably faster than complex PSF shape analysis and relatively insensitive to geometrical aberrations. We demonstrate that 4polar3D can resolve nanoscale molecular organization in whole cells' crowded structures, uncovering 3D-oriented actin filament networks in densely packed lamellipodia and podosomes.