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Multiview light-sheet microscope for rapid in toto imaging

Abstract

We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biological specimens with subcellular resolution. The fixed geometrical arrangement of the imaging branches enables multiview data fusion in real time. The high speed of MuVi-SPIM allows faithful tracking of nuclei and cell shape changes, which we demonstrate through in toto imaging of the embryonic development of Drosophila melanogaster.

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Figure 1: MuVi-SPIM microscope setup and data processing pipeline.
Figure 2: Rapid and reliable MuVi-SPIM imaging of highly dynamic developmental processes.

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Acknowledgements

We thank the mechanical and electronics workshop of the European Molecular Biology Laboratory (EMBL) for customized hardware; D. Holzer, T. Schneidt and H. Gustafson for help with the flies and G. Heuvelman and M. Wachsmuth for discussions on optics. We thank S. DeRenzis and the Wieschaus laboratory for the Gap43-mCherry flies and E. Lemke for hardware support. We thank J. Ellenberg for his helpful comments on the manuscript and anonymous reviewers for their constructive comments. We acknowledge the advanced light microscopy facility of EMBL for its kind support. We are grateful for financial support from EMBL, the EMBL Interdisciplinary Postdocs Program (EIPOD) and the Center for Modelling and Simulation in the Biosciences (BIOMS).

Author information

Authors and Affiliations

Authors

Contributions

U.K. and L.H. designed and built the microscope software and hardware. U.K., S.G. and T.E.S. performed the experiments. U.K., S.G., T.E.S. and S.J.S. performed data analysis, quantification and visualization. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Lars Hufnagel.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Notes 1–6 and Supplementary Protocol (PDF 5595 kb)

Rotation of a background-corrected raw His2Av-mCherry–tagged embryo in cycle 11

Color codes for depth (blue-labeled nuclei are on one side; red-labeled nuclei are on the other side). (MOV 5010 kb)

Fused raw data of an embryo expressing His2Av-mCherry for a single time point in cycle 13

Depth inside the embryo is indicated to the left. Adjacent images in the 3D stack were taken 2 μm apart from each other. Anterior (A)–posterior (P) and dorsal (D)–ventral (V) axes of the embryo are indicated in the top-right corner. (MOV 1396 kb)

A view on the raw data from Supplementary Video 2 along the embryo's long axis, shown from the anterior to the posterior pole

Slice spacing is 0.5 μm. The dorsal (D)–ventral (V) axis is indicated. (MOV 5670 kb)

Anaglyphic 3D representation of the image from Supplementary Video 2

The image was background-corrected and processed for red-cyan anaglyph glasses. (MOV 10096 kb)

Dynamics of nuclear motion. Positions of the segmented nuclei are shown at time T (indicated in top right corner) and ten preceding time points

Color ranges from blue to yellow, where the former corresponds to the time T, and the latter to T – 250 s. (MOV 51682 kb)

Tracking of nuclei

Each point represents a tracked nucleus. After division, one of the daughter nuclei is assigned the mother's color and the other nucleus is assigned a new unique color. Scale bar, 70 μm. (MOV 21002 kb)

Velocity of nuclei

Each point represents a nucleus. Nucleus position is shown for the current and previous four time points. The color indicates velocity of each nucleus (see color bar at the bottom). Scale bar, 70 μm. (MOV 6735 kb)

Ventral and the dorsal (inset) views of a Drosophila embryo starting from early cycle 14

The embryo was imaged for 15 h every 30 s, and for a further 5 h every 10 min to confirm that the high frame rate of imaging didn't alter the embryonic development. Only one image every 10 min is shown in the video after the initial 5 h. Every 3D image was background-corrected and maximum-projected from a middle plane towards the ventral and dorsal side, respectively, using Fiji (www.fiji.sc). (MOV 14452 kb)

Six nuclei, which become part of the ventral ectoderm from the recording shown in Supplementary Video 8, were tracked through several hours of embryogenesis

The left panel shows tracked color-coded nuclei (parent and daughter nuclei have the same color) with path traces. The right panel shows a magnified region around the five leftmost nuclei. Each colored dot marks the brightest pixel of the appropriate nucleus. Note that this video is a 2D projection of the data, but the trajectories are tracked in three dimensions. Hence, sometimes nuclei appear to intersect, but in three dimensions the nuclei move past each other without collision. (MOV 23285 kb)

Rotation of a Drosophila melanogaster embryo expressing the Gap43-mCherry membrane marker in cycle 14, corresponding to Figure 2e

Rotation of a Drosophila melanogaster embryo expressing the Gap43-mCherry membrane marker in cycle 14, corresponding to Figure 2e. (MOV 32144 kb)

Four concurrently running perpendicular sections through a Gap43-mCherry membrane marker–expressing embryo during cycle 14

Top panels are the opposing lateral views, bottom left is ventral view and bottom right is dorsal view. This video corresponds to Supplementary Figure 9c. (MOV 2694 kb)

View along the long-axis of a Drosophila melanogaster embryo expressing the Gap43-mCherry membrane marker at a fixed time point, 10 min prior to ventral furrow formation

View along the long-axis of a Drosophila melanogaster embryo expressing the Gap43-mCherry membrane marker at a fixed time point, 10 min prior to ventral furrow formation. (MOV 46257 kb)

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Krzic, U., Gunther, S., Saunders, T. et al. Multiview light-sheet microscope for rapid in toto imaging. Nat Methods 9, 730–733 (2012). https://doi.org/10.1038/nmeth.2064

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