Biological Sciences Division
Published February 21, 2025 | Version v1
Journal article Open

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

Creators

  • 1. University of Chicago
  • 2. Zhejiang University
  • 3. National Institute of Biomedical Imaging and Bioengineering
  • 4. Virginia Tech
  • 5. National Heart, Lung, and Blood Institute
  • 6. Marine Biological Laboratory
  • 7. University of Massachusetts
  • 8. Simon Fraser University
  • 9. Lund University

Description

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.

Data availability

Multidimensional images, preprocessing analysis software, and reconstruction and visualization software data have been deposited in BioImage Archive (https://www.ebi.ac.uk/biostudies/bioimages/studies/S-BIAD1055) (62) and GitHub (https://github.com/eguomin/microImageLib (63); https://github.com/talonchandler/polaris (64)).

Files

chandler-et-al-2025-volumetric-imaging-of-the-3d-orientation-of-cellular-structures-with-a-polarized-fluorescence-light.pdf

Files (106.3 MB)

Additional details

Identifiers

DOI
10.1073/pnas.2406679122
Other
oai:uchicago.tind.io:14623

Funding

Whitman Fellows program
National Institutes of Health
R01EB026300
Gordon and Betty Moore Foundation
HHMI and the Janelia Visiting Scientists Program
Chan Zuckerberg Initiative
Arnold and Mabel Beckman Foundation
U.S. Department of Energy
DE-FG-03ER15421
National Institute of General Medical Sciences
R35GM131843
Marine Biological Laboratory
Inoué Endowment Fund
National Science Foundation
1762468
National Science Foundation
2119949
National Science Foundation
2422340
National Institutes Health
1R01 HL162822-01A1
Virginia Tech
National Natural Science Foundation of China
62427807
National Natural Science Foundation of China
62475232
Zhejiang University
Global Partnership Fund

UChicago Information

Division(s)
Biological Sciences Division
Department(s)
Radiology