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Comment
. 2025 Apr 8;122(14):e2501914122.
doi: 10.1073/pnas.2501914122. Epub 2025 Mar 31.

Painting rich six-dimensional pictures using polarized fluorescence microscopy

Matthew D Lew  1
Affiliations
Comment

Painting rich six-dimensional pictures using polarized fluorescence microscopy

Matthew D Lew. Proc Natl Acad Sci U S A. .
No abstract available

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Conflict of interest statement

Competing interests statement:The author declares no competing interest.

Figures

Fig. 1.
Fig. 1.
Principles of polarized dual-view inverted selective-plane illumination microscopy (pol-diSPIM). (A) Any set of molecular orientations may be represented as a distribution fθ,ϕ on a unit hemisphere. Red: Dipoles aligned at θ=45∘,ϕ=0∘, purple: dipoles aligned at θ=45∘,ϕ=180∘, blue: dipoles uniformly distributed within a cone of solid angle Ω=2π/3 at a peak orientation θ=45∘,ϕ=0∘. (B) Normalized angular spectra Flm corresponding to orientation distributions in (A), where fθ,ϕ=∑l,mFlmYlmθ,ϕ. Any polarized fluorescence microscope that measures solely absorption or emission transition dipole moments is capable of measuring only F00,F20,F21,F2-1,F22, and F2-2, which is termed the angular diffraction limit (11). (C) Spherical harmonics Ylmθ,ϕ plotted on the unit hemisphere corresponding to (A). (D) Schematic of pol-diSPIM with detection and illumination objective lenses; the roles of these lenses alternate during data collection. Three tilted and polarized light sheets (green) are shown with polarization vectors E1,E2, and E3, which sequentially excite three distributions of dipoles, corresponding to (A). Gray line: optical axis of detection objective. (E) Simulated raw pol-diSPIM images of orientation distributions shown in (A), collected using tilted and polarized light sheets in (D). Top: green bar depicts path of beam E1 and the focal plane of the detection objective. The leftmost three sets of dipoles are in focus, while the far-right set of red dipoles is defocused by 300 nm. Note that E1 produces images of the red and purple distributions that are identical to one another and are also extremely similar to that of the blue cone distribution. Only after collecting images using tilted light sheets E2 and E3 can pol-diSPIM easily distinguish between these distributions. Defocused and aligned dipoles (red dipoles, 4th column) produce similar images to those of in-focus and wobbling dipoles (blue cone, 3rd column), illustrating spatio-angular coupling. Colorbars: (C) normalized amplitude, (E) normalized intensity. (Scalebar: 500 nm.)
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References

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