Abstract
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.
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Data availability
The 4polar3D raw image stacks generated in this study are available upon request from the corresponding author (due to the large size of the files, 150-250 GB). A subset of data (2 GB), as well as the processed orientation/detection parameters from single-molecule data generated in this study are available in the Source Data file (https://doi.org/10.6084/m9.figshare.28890470). Processed data for example ROIs generated in this study are available for download at https://github.com/CessVala/4polar3D_SMOLM (TestData folder), with explanations provided in the README.md file.
Code availability
The MATLAB code used to analyze the data (The MathWorks, Inc. Recommended version: R2020a or newer) is available on GitHub at https://github.com/CessVala/4polar3D_SMOLM (https://doi.org/10.5281/zenodo.18663231), which includes a manual and installation instructions. The Python code used for the Monte Carlo simulation (Python Software Foundation. Recommended version, Python 3.8 or newer) is available on GitHub at https://github.com/CessVala/4polar3D_SMOLM_simulation (https://doi.org/10.5281/zenodo.18663511).
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Acknowledgements
The authors warmly thank Simli Dey and Feng-Ching Tsai (Institut Curie, Paris) for the preparation of the Silica beads coated with lipid bilayers. This research has received funding from the France 2030 investment plan managed by the PIA France 2030 program IDEC Equipex+ grant (ANR-21-ESRE-0002) and the Initiative d’Excellence d’Aix-Marseille Université (S.B.) - A*MIDEX Institutes Cancer et Immunologie (AMX-19-IET-001) and Marseille Imaging (C.S.S.-K.). This work is also funded by the ANR grants 3DPolariSR (ANR-20-CE42-0003) (S.B.) and SETIPSS (ANR-22-CE13-0039) (S.B., M.V.), the « Investissements d’Avenir » program managed by the ANR (ANR-16-CONV-0001) (S.B.), France BioImaging national infrastructure ANR-10-INBS-04-07 (S.B.), and from CNRS (S.B.). This work was also funded by the Chinese Academy of Sciences President’s International Fellowship Initiative grant 2024FSB0003 (C.A.V.-C.).
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C.S.S.-K. and M.S. co-developed the experimental setup, performed experiments and wrote data analysis codes. C.A.V.-C. developed the data processing and Monte Carlo simulation codes. L.A.A.-C. developed simulations on CRLB calculations. V.C. developed the initial version of the experimental setup. J.R.B and R.P prepared the cells dedicated to podosomes studies. M.M. supervised the cells preparations. M.A.A. contributed to data analysis decisions. S.B. conceived the project, developed simulations and wrote the manuscript. All authors contributed to feedbacks and edits on the manuscript.
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The 4polar3D microscope was invented by S.B. and V.C. and is covered by US patent 11927737 (2022), which was filed by and assigned to Université Aix Marseille, Centre national de la recherche scientifique and Centrale Marseille.
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Senthil Kumar, C.S., Valades Cruz, C.A., Sison, M. et al. 4polar3D single molecule imaging of 3D orientation in dense actin networks using ratiometric polarization splitting. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70852-y
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DOI: https://doi.org/10.1038/s41467-026-70852-y
