Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations in biological samples, 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 diffraction-limited three-dimensional distribution of the orientations and positions of ensembles of fluorescent dipoles that label biological structures. We share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model the distributions based on the polarization-dependent efficiency of excitation and detection of emitted fluorescence, 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-labeled giant unilamellar vesicles, fast-scarlet-labeled cellulose in xylem cells, and phalloidin-labeled actin in U2OS cells. Additionally, we observe phalloidin-labeled actin in mouse fibroblasts grown on grids of labeled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

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Publication Details

Journal
Proceedings of the National Academy of Sciences
Published
2025-02-21
DOI
https://doi.org/10.1073/pnas.2406679122
Citations
6
Primary Topic
Advanced Fluorescence Microscopy Techniques
Type
article
Field-Weighted Citation Impact
9.80

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article

Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope

Valentin Jaumouillé, Rudolf Oldenbourg, Tobias I. Baskin, Min Yi Guo et al.
6 citations
Proceedings of the National Academy of Sciences
Advanced Fluorescence Microscopy Techniques
9.80
article

Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope

Valentin Jaumouillé, Rudolf Oldenbourg, Tobias I. Baskin, Min Yi Guo, Shalin B. Mehta, Abhishek Kumar, Talon Chandler, Robert S Fischer, Atharva Agashe, Vinay Swaminathan, Jiji Chen, Hari Shroff, Yicong Wu, Yijun Su, Patrick J. La Rivière, Amrinder S. Nain, Huafeng Liu, Junyu Liu
article en
6 citations

Abstract

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations in biological samples, 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 diffraction-limited three-dimensional distribution of the orientations and positions of ensembles of fluorescent dipoles that label biological structures. We share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model the distributions based on the polarization-dependent efficiency of excitation and detection of emitted fluorescence, 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-labeled giant unilamellar vesicles, fast-scarlet-labeled cellulose in xylem cells, and phalloidin-labeled actin in U2OS cells. Additionally, we observe phalloidin-labeled actin in mouse fibroblasts grown on grids of labeled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

Proceedings of the National Academy of SciencesVol. 122(8)
Scania (Sweden) (SE), Marine Biological Laboratory (US), Bell (Canada) (CA), Simon Fraser University (CA), Lund University (SE), Janelia Research Campus (US), University of Massachusetts Amherst (US), University of Chicago (US), National Institute of Biomedical Imaging and Bioengineering (US), Key-Whitman Eye Center (US), National Heart, Lung, and Blood Institute (US), Chan Zuckerberg Initiative (United States) (US), Advanced Imaging Research (United States) (US), Optica (US), Zhejiang University (CN), Virginia Tech (US)
National Science Foundation, U.S. Department of Energy, National Natural Science Foundation of China, National Institutes of Health, National Institute of General Medical Sciences
Openalex Percentile: Top 2%
Advanced Fluorescence Microscopy Techniques
9.80
DOIOpen Access PDFOpenAlex
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