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Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy


Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.

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Figure 1: Dual-view iSPIM setup.
Figure 2: Improving resolution isotropy with different fusion schemes.
Figure 3: Bleaching comparison between SDCM and diSPIM.
Figure 4: Dual-view iSPIM enables microtubule tracking in 4D.
Figure 5: Dual-view iSPIM improves axial resolution in 4D embryonic imaging.
Figure 6: Spatiotemporal dissection of AIY neurite outgrowth.


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We thank J. McNally and C.-H. Lee for illuminating discussions, M.R. Reinhardt for helping us with the scientific cMOS, W. Mohler for helping us to share and view 4D data sets, G. Rondeau for help with mechanical design, A. Hoofring for help with illustrations and H. Eden for critical feedback on the manuscript. A.S. and Z.B. acknowledge funding from National Institutes of Health (NIH) grants GM097576 and HD075602. R.C. and D.C.-R. acknowledge funding from US National Institutes of Health (NIH) grants R01 NS076558 and U01HD075602. This work was supported by the Intramural Research Programs of the NIH National Institute of Biomedical Imaging and Bioengineering, the National Institute of Heart, Lung, and Blood, and the Center for Information Technology.

Author information

Authors and Affiliations



Conceived idea and supervised project: H.S. Designed optical system: Y.W. and H.S. Built optical system: Y.W. and P.W.W. Took data and prepared samples: Y.W., P.W. and R.S.F. Implemented joint deconvolution algorithm: A.G.Y. and Y.W. Developed rotation and registration algorithms: J.S. and M.M. Provided guidance on nematode experiments: R.C., A.S., Z.B. and D.A.C.-R. Provided guidance on microtubule experiments: R.S.F. and C.M.W. Provided reagents and materials: R.C., A.S., Z.B., D.A.C.-R., R.S.F., and C.M.W. Analyzed data: Y.W., P.W., R.S.F., R.C., A.S., C.M.W., Z.B., D.A.C.-R. and H.S. Wrote paper: Y.W., J.S., R.S.F., R.C., C.M.W., D.A.C.-R. and H.S.

Corresponding author

Correspondence to Yicong Wu.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Note and Supplementary Table 1 (PDF 1551 kb)

Supplementary Video 1

Comparison of subdiffractive beads with different fusion schemes. (AVI 249 kb)

Supplementary Video 2

Comparison between SDCM and diSPIM on GFP-EB3 microtubules in live human umbilical vein endothelial cells. (AVI 2400 kb)

Supplementary Video 3

Comparison of 3D GFP-EB3 microtubule dynamics in human umbilical vein endothelial cells of different thickness and in different cellular environments with diSPIM. (AVI 4162 kb)

Supplementary Video 4

SDCM volumetric time series of GFP-histones in live, BV24 nematode embryos. (AVI 10074 kb)

Supplementary Video 5

Comparative iSPIM and diSPIM volumetric time series of GFPhistones in a live BV24 nematode embryo from the 4 cell stage up to hatching. (AVI 26620 kb)

Supplementary Video 6

Comparison between iSPIM and diSPIM, when visualizing neuronal processes in developing embryo. (AVI 11921 kb)

Supplementary Video 7

Comparison between iSPIM and diSPIM, highlighting differences in a single volume with GFP-labeled AIY neurons. (AVI 507 kb)

Supplementary Video 8

DiSPIM enables visualization of AIY outgrowth processes in a live DCR553 nematode embryo. (AVI 32825 kb)

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

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