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

Chandler, T.; Guo, M.; Su, Y.; Chen, J.; Wu, Y.; Liu, J.; Agashe, A.; Fischer, R. S.; Mehta, S. B.; Kumar, A.; Baskin, T. I.; Jamouille, V.; Liu, H.; Swaminathan, V.; Nain, A.; Oldenbourg, R.; La Riviere, P.; Shroff, H.

doi:10.1101/2024.03.09.584243

bioRxiv · 10.1101/2024.03.09.584243

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

Chandler, T.·Guo, M.·Su, Y.·Chen, J.·Wu, Y.·Liu, J.·Agashe, A.·Fischer, R. S.·Mehta, S. B.·Kumar, A.·Baskin, T. I.·Jamouille, V.·Liu, H.·Swaminathan, V.·Nain, A.·Oldenbourg, R.·La Riviere, P.·Shroff, H.

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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.

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Chandler, T., Guo, M., Su, Y., Chen, J., Wu, Y., Liu, J., Agashe, A., Fischer, R. S., Mehta, S. B., Kumar, A., Baskin, T. I., Jamouille, V., Liu, H., Swaminathan, V., Nain, A., Oldenbourg, R., La Riviere, P., Shroff, H.. 2024-03-12. Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM). https://doi.org/10.1101/2024.03.09.584243

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