Fluorescence Interference-Based Polarized Structured Illumination Microscopy for High Axial Accuracy Morphology Imaging of Dipole Orientations

The dipole orientation of fluorescent molecules reveals the structural organization at the subcellular level, and imaging its distribution is important for fundamental cell biology studies. Using conventional fluorescence microscopy, the orientation of fluorescent dipoles can be obtained by demodulating the polarization states through excitation or emission processes. The recent introduction of structured illumination microscopy (SIM) has improved the axial and lateral resolution to around 300 and 100 nm, respectively. However, the axial resolution is still very limited, which hinders the capabilities to offer more precise measurements of polarization information. Here, we report Fluorescence Interference based polarized structured illumination microscopy (FI-pSIM), which provides a sub-30 nm axial reconstruction accuracy and maintains the same lateral resolution comparable to SIM by only 9 raw images. By synergizing the fluorescence interference via the 4Pi configuration and the polarization demodulation of structured illumination, FI-pSIM enables three-dimensional morphological imaging of ensemble dipole orientations in subcellular structures with ∼ sub-30 nm accuracy of axial reconstruction. The high efficacy and four-dimensional (4D) imaging modality of FI-pSIM are demonstrated by mapping the distribution and orientation of fluorescent dipoles in biological microfilaments. By elucidating the dipole orientations, we envision FI-pSIM may open up new avenues for demystifying the 4D organizations of subcellular structures.