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Whole-animal functional and developmental imaging with isotropic spatial resolution

Nature Methods volume 12, pages 11711178 (2015) | Download Citation

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Abstract

Imaging fast cellular dynamics across large specimens requires high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To meet these requirements, we developed isotropic multiview (IsoView) light-sheet microscopy, which rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. Combining these four views by means of high-throughput multiview deconvolution yields images with high resolution in all three dimensions. We demonstrate whole-animal functional imaging of Drosophila larvae at a spatial resolution of 1.1-2.5 μm and temporal resolution of 2 Hz for several hours. We also present spatially isotropic whole-brain functional imaging in Danio rerio larvae and spatially isotropic multicolor imaging of fast cellular dynamics across gastrulating Drosophila embryos. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

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Change history

  • 05 November 2015

    In the version of this article initially published online, Supplementary Video 5 was incorrectly labeled. The error has been corrected as of 5 November 2015.

References

  1. 1.

    & Faster fluorescence microscopy: advances in high speed biological imaging. Curr. Opin. Chem. Biol. 20, 46–53 (2014).

  2. 2.

    & Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy. Neuron 85, 462–483 (2015).

  3. 3.

    et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

  4. 4.

    et al. Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013).

  5. 5.

    et al. Whole-central nervous system functional imaging in larval Drosophila. Nat. Commun. 6, 7924 (2015).

  6. 6.

    , , & Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nat. Methods 9, 755–763 (2012).

  7. 7.

    , , , & Multiview light-sheet microscope for rapid in toto imaging. Nat. Methods 9, 730–733 (2012).

  8. 8.

    et al. High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics. Nat. Commun. 4, 2207 (2013).

  9. 9.

    et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

  10. 10.

    et al. Light-sheet functional imaging in fictively behaving zebrafish. Nat. Methods 11, 883–884 (2014).

  11. 11.

    et al. High-resolution reconstruction of the beating zebrafish heart. Nat. Methods 11, 919–922 (2014).

  12. 12.

    et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Nat. Photonics 9, 113–119 (2015).

  13. 13.

    , , , & Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).

  14. 14.

    et al. Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy. Front. Neural Circuits 7, 65 (2013).

  15. 15.

    et al. Whole-brain functional imaging with two-photon light-sheet microscopy. Nat. Methods 12, 379–380 (2015).

  16. 16.

    , , , & Multi-view image fusion improves resolution in three-dimensional microscopy. Opt. Express 15, 8029–8042 (2007).

  17. 17.

    et al. Efficient Bayesian-based multiview deconvolution. Nat. Methods 11, 645–648 (2014).

  18. 18.

    , & Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993).

  19. 19.

    , , & Thin laser light sheet microscope for microbial oceanography. Opt. Express 10, 145–154 (2002).

  20. 20.

    , , , & Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

  21. 21.

    , , & Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).

  22. 22.

    & Scanned light sheet microscopy with confocal slit detection. Opt. Express 20, 21805–21814 (2012).

  23. 23.

    , , & Software for bead-based registration of selective plane illumination microscopy data. Nat. Methods 7, 418–419 (2010).

  24. 24.

    , , , & Spatially-variant Lucy-Richardson deconvolution for multiview fusion of microscopic 3D images. In Proc. IEEE International Symposium on Biomedical Imaging 899–904 (IEEE, 2011).

  25. 25.

    et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

  26. 26.

    et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).

  27. 27.

    , , & A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. USA 98, 15050–15055 (2001).

  28. 28.

    & Random sample consensus—a paradigm for model-fitting with applications to image-analysis and automated cartography. Commun. ACM 24, 381–395 (1981).

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Acknowledgements

We thank B. Coop and the Janelia ID&F Team for mechanical designs and custom mechanical parts; M. Coleman for custom microscope-operating software; V. Stamataki for help with bead injections in live Drosophila embryos and larvae; E. Betzig, K. Branson and G. Rubin for helpful discussions; M. Ahrens and C.-T. Yang (Janelia Research Campus, Ashburn, Virginia, USA) for the GCaMP6s zebrafish line; E. Wieschaus (Howard Hughes Medical Institute, Princeton University, Princeton, New Jersey, USA) for the Spider-GFP flies; the Janelia Fly Facility (Janelia Research Campus, Ashburn, Virginia, USA) for sharing and maintaining transgenic Drosophila stocks; and the Janelia Vivarium Team for zebrafish animal care. This work was supported by the Howard Hughes Medical Institute.

Author information

Affiliations

  1. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.

    • Raghav K Chhetri
    • , Fernando Amat
    • , Yinan Wan
    • , Burkhard Höckendorf
    • , William C Lemon
    •  & Philipp J Keller

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Contributions

P.J.K. and R.K.C. designed the IsoView microscope. R.K.C. built the IsoView microscope and performed all imaging experiments. F.A. developed the IsoView image-processing framework. Y.W. and W.C.L. prepared Drosophila specimens for IsoView imaging. B.H. prepared zebrafish specimens for IsoView imaging. P.J.K. conceived of the research, supervised the project and wrote the paper, with contributions from R.K.C.

Competing interests

P.J.K. filed a provisional US patent application for IsoView microscopy on October 9, 2013, and P.J.K. and R.K.C. filed a nonprovisional US patent application on October 8, 2014 (application number 14/509,331).

Corresponding authors

Correspondence to Raghav K Chhetri or Philipp J Keller.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Tables 1 and 2, and Supplementary Note 1

Zip files

  1. 1.

    Supplementary Data 1

    Technical drawings of IsoView custom mechanical components

  2. 2.

    Supplementary Data 2

    Zemax models of IsoView custom optics

Videos

  1. 1.

    Animation of the IsoView microscope

    The video shows an animation of the IsoView microscope model. Additional information about IsoView microscope design is provided in the Supplementary Materials, including parts and assembly drawings for all mechanical and optical components of the microscope (Supplementary Data 1), Zemax models of all custom optical components (Supplementary Data 2), a microscope parts list including electronics and computational hardware (Supplementary Table 1, Online Methods), and instructions for setting up and aligning the IsoView microscope (Supplementary Note, Supplementary Figures 1, 2, 3, 4, 5). Note: The video is provided in DivX format. A video player and codecs for the DivX format are freely available at http://www.divx.com/en/software/divx.

  2. 2.

    Comparison of conventional and IsoView functional imaging

    Rotating maximum-intensity projections of conventional, non-isotropic image data (left and middle panels) and IsoView image data (right panel) of a stage 17 Drosophila embryo expressing the calcium indicator GCaMP6s throughout the nervous system. The first two panels show pairwise combinations of the four raw, non-isotropic views recorded by IsoView (left: 0° and 180° combined views, middle: 90° and 270° combined views). Views recorded along the same optical axis were fused by adaptive blending, following the standard image processing workflow used for SiMView image data sets (Tomer et al. 2012, Nature Methods), and represent spatial resolution and image quality obtained with conventional light-sheet microscopy. The horizontal dimensions along which spatial resolution is high and low, respectively, are flipped in orthogonal views. The panel to the right shows the final, multi-view deconvolved IsoView image data, which combines information from all four views. Spatial resolution as well as resolution isotropy are greatly improved by orthogonal four-view imaging in conjunction with multi-view image deconvolution. Image data were acquired in IsoView mode 2 (simultaneous four-view imaging with phase-shifted confocal detection, see Fig. 1c) and are displayed using gamma correction and a false-color look-up-table (blue to yellow) to reduce the high dynamic range of the raw data (average signal-to-noise ratio: 211 ± 76, mean ± SD, n = 16) for better visibility. Note: The video is provided in DivX format. A video player and codecs for the DivX format are freely available at http://www.divx.com/en/software/divx.

  3. 3.

    IsoView whole-animal functional imaging in larval Drosophila

    Lateral (left), dorsoventral (middle) and rotating (right) maximum-intensity projections of a multi-view deconvolved IsoView time-lapse recording of an early Drosophila first instar larva expressing the calcium indicator GCaMP6s throughout the nervous system. Functional imaging was performed at 2 Hz in IsoView mode 1 (pairwise sequential four-view imaging). The images are gamma-corrected and shown using a false-color look-up-table (blue to yellow) to reduce the high dynamic range of the raw data for better visibility. Note: The video is provided in DivX format. A video player and codecs for the DivX format are freely available at http://www.divx.com/en/software/divx.

  4. 4.

    Long-term IsoView whole-animal functional imaging

    Dorsoventral (top) and lateral (bottom) maximum-intensity projections of an IsoView long-term time-lapse recording of a stage 17 Drosophila embryo expressing the calcium indicator GCaMP6s throughout the nervous system. The embryo develops into a first instar larva that crawls out of the imaging volume at the end of the recording. Imaging was performed at 2 Hz in IsoView mode 1 (pairwise sequential four-view imaging) over a period of 9 hours, by alternating between 30-min imaging sessions and 30-min breaks to maximize experiment duration while conserving disk space. Over 120,000 single-view image volumes (4.5 million images, 9 terabytes) were acquired for this specimen, resulting in more than 30,000 multi-view deconvolved high-resolution IsoView image stacks. Note: The video is provided in DivX format. A video player and codecs for the DivX format are freely available at http://www.divx.com/en/software/divx.

  5. 5.

    IsoView whole-brain functional imaging in larval zebrafish.

    Dorsoventral (left) and rotating (right) maximum-intensity projections of a multi-view deconvolved IsoView time-lapse recording of the brain of a 3-day old larval zebrafish expressing the calcium indicator GCaMP6s throughout its nervous system. Functional imaging was performed at 1 Hz in IsoView mode 1 (pairwise sequential four-view imaging). The video captures several instances of motor-related, large-scale brain activity in the awake animal. For better visibility of the large dynamic range of the image data, images are presented using a false-color look-up-table (blue to yellow). The video is provided in DivX format. A video player and codecs for the DivX format are freely available at http://www.divx.com/en/software/divx.

  6. 6.

    Comparison of conventional and IsoView developmental imaging

    Rotating maximum-intensity projections of conventional, non-isotropic image data (left and middle panels) and IsoView image data (right panel) of a stage 5 Drosophila embryo ubiquitously expressing GFP targeted to membranes and RFP targeted to cell nuclei (colors are inverted for better visibility). The first two panels show pairwise combinations of the four raw, non-isotropic views recorded by IsoView (left: 0° and 180° combined views, middle: 90° and 270° combined views). Views recorded along the same optical axis were fused by adaptive blending, following the standard image processing workflow used for SiMView image data sets (Tomer et al. 2012, Nature Methods), and represent spatial resolution and image quality obtained with conventional light-sheet microscopy. The horizontal dimensions along which spatial resolution is high and low, respectively, are flipped in orthogonal views. The panel to the right shows the final, multi-view deconvolved IsoView image data, which combines information from all four views. Spatial resolution as well as resolution isotropy are greatly improved by orthogonal four-view imaging in conjunction with multi-view image deconvolution. Image data were acquired in IsoView modes 2 and 3 (four-view two-color imaging with phase-shifted confocal detection, see Fig. 1c,d) and are displayed using gamma correction to reduce the high dynamic range of the raw data (average signal-to-noise ratio: 132 ± 43, mean ± SD, n = 8) for better visibility. We note that the vertical transition regions visible in the pairwise combined views (at 0° and 90° rotation angles for the middle and left panels, respectively) are a result of linear blending of the two image volumes and imperfect physical coverage of these regions by two-view imaging. Owing to the two-fold better physical coverage of IsoView four-view imaging, such imaging artifacts are avoided in the IsoView reconstruction (right panel). Note: The video is provided in DivX format. A video player and codecs for the DivX format are freely available at http://www.divx.com/en/software/divx.

  7. 7.

    IsoView two-color imaging of Drosophila gastrulation

    From left to right: dorsal, ventral, lateral-left, lateral-right and rotating maximum-intensity projections of a multi-view deconvolved IsoView time-lapse recording of a Drosophila embryo ubiquitously expressing GFP targeted to membranes and RFP targeted to cell nuclei (colors are inverted for better visibility). The video shows embryonic development during stages 6-8. Image data were acquired at 4-second intervals in IsoView modes 2 and 3 (four-view two-color imaging with phase-shifted confocal detection, see Fig. 1c,d) and are displayed using gamma correction to reduce the high dynamic range of the raw data for better visibility. Note: The video is provided in DivX format. A video player and codecs for the DivX format are freely available at http://www.divx.com/en/software/divx.

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DOI

https://doi.org/10.1038/nmeth.3632

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