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

• 成果类型: Article
• 署名作者: Chandler, Talon; Guo, Min; Su, Yijun; Chen, Jiji; Wu, Yicong; Liu, Junyu; Agashe, Atharva; Fischer, Robert S.; Mehta, Shalin B.; Kumar, Abhishek; Baskin, Tobias I.; Jaumouille, Valentin; Liu, Huafeng; Swaminathan, Vinay; Nain, Amrinder S.; Oldenbourg, Rudolf; La Riviere, Patrick J.; Shroff, Hari
• 署名单位: Chan Zuckerberg Initiative (CZI); University of Chicago; Zhejiang University; National Institutes of Health (NIH) - USA; NIH National Institute of Biomedical Imaging & Bioengineering (NIBIB); National Institutes of Health (NIH) - USA; Howard Hughes Medical Institute; Virginia Polytechnic Institute & State University; National Institutes of Health (NIH) - USA; NIH National Heart Lung & Blood Institute (NHLBI); Marine Biological Laboratory - Woods Hole; University of Massachusetts System; University of Massachusetts Amherst; Marine Biological Laboratory - Woods Hole; Simon Fraser University; Scania; Lund University; Lund University; Scania
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14808
• DOI: 10.1073/pnas.2406679122
• 发表日期: 2025-02-21
• 关键词: living cells; excitation; diffusion; migration; DYNAMICS; BEHAVIOR; ORGANIZATION; collagen; shape

摘要

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 twodimensional 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.