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Main Menu - Block
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Invertebrate Shared Resource
- Janelia Experimental Technology
- Mass Spectrometry
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
- Scientific Computing Software
- Scientific Computing Systems
- Viral Tools
- Vivarium
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.
