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

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

Ngôn ngữ: eng

Ký hiệu phân loại: 664.725 Rice

Thông tin xuất bản: United States : Proceedings of the National Academy of Sciences of the United States of America , 2025

Mô tả vật lý:

Bộ sưu tập: NCBI

ID: 250360

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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