Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)

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bioRxiv. 2024 Mar 12;. doi: 10.1101/2024.03.09.584243

Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM) Shroff Lab

Chandler Talon, Guo Min, Su Yijun, Chen Jiji, Wu Yicong, Liu Junyu, Agashe Atharva, Fischer Robert, Mehta Shalin, Kumar Abhishek, Baskin Tobias, Jamouille Valentin, Liu Huafeng, Swaminathan Vinay, Nain Amrinder, Oldenbourg Rudolf, La Riviere Patrick, Shroff Hari

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Abstract

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.Competing Interest StatementH.S., A.K., S.M., P.L.R., R.O., Y.W., and T.C. hold US Patent #11428632.

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bioXiv preprint https://doi.org/10.1101/2024.03.09.584243

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Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)

Abstract

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