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Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)
- PMID: 38712306
- PMCID: PMC11071302
- 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)
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.
Keywords: inverse problems; light-sheet fluorescence microscopy; molecular orientation; polarization.
Conflict of interest statement
Conflict of interest/Competing interests. H. S., A. K., S. M., P. L. R., R. O., Y. W., and T. C. hold US Patent # 11428632. Ethics approval and consent to participate. Not applicable.
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