Resolution doubling in light-sheet microscopy via oblique plane structured illumination

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluores...

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Bibliographic Details
Published inNature methods Vol. 19; no. 11; pp. 1419 - 1426
Main Authors Chen, Bingying, Chang, Bo-Jui, Roudot, Philippe, Zhou, Felix, Sapoznik, Etai, Marlar-Pavey, Madeleine, Hayes, James B., Brown, Peter T., Zeng, Chih-Wei, Lambert, Talley, Friedman, Jonathan R., Zhang, Chun-Li, Burnette, Dylan T., Shepherd, Douglas P., Dean, Kevin M., Fiolka, Reto P.
Format Journal Article
LanguageEnglish
Published New York Nature Publishing Group US 01.11.2022
Nature Publishing Group
Subjects
631/1647/328/2237
631/1647/328/2238
Bioinformatics
Biological Microscopy
Biological Techniques
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Chemical compounds
Excitation
Fluorescence
Fluorescence microscopy
Fluorophores
Illumination
Imaging
Imaging, Three-Dimensional - methods
Laser microscopy
Life Sciences
Light
Light sheets
Lighting
Microscopes
Microscopy
Microscopy, Fluorescence - methods
Optical sectioning
Photobleaching
Photochemical reactions
Phototoxicity
Proteomics
Resolution
Sectioning
Spatial discrimination
Spatial resolution
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Summary:Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz. The longstanding goal of combining the optical sectioning of light-sheet illumination and the resolving power of multidirectional structured illumination microscopy is realized using an oblique plane microscope for improved fast 3D subcellular imaging.
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Author Contributions
These authors have contributed equally to the manuscript
R.F. conceived the idea of OPSIM and the image rotator. B.C. mathematically described the image rotator and B.J.C performed numerical simulations and practical experiments. B.C., B.J.C. and R.F. designed an experimentally tractable image rotator. B.C build the image rotator. R.F. build the microscope. R.F. and B.J.C. acquired experimental data. R.F., P.B., B.J.C. and B.C. performed data processing. P.R. and F.Z. wrote the fine registration algorithm. R.F., B.J.C, P.B and D.S. wrote the SIM reconstruction software. M.M., J.H., J.F., E.S., K.D.M., D.B., C.W.Z. and C.L.Z provided biological samples. E.S. and J.H. performed imaging with the Sora and iSIM system, respectively.
ISSN:1548-7091
1548-7105
DOI:10.1038/s41592-022-01635-8