OpenSPIM: an open-access light-sheet microscopy platform (original) (raw)

To the Editor:

Light-sheet microscopy is revolutionizing biology by enabling live in toto imaging of entire embryos or organs with minimal phototoxicity1. We present an open hardware and software platform for constructing a customizable microscope for selective-plane illumination microscopy (SPIM). The OpenSPIM platform is shared with the scientific community through a public website (http://openspim.org/), making light-sheet microscopy more accessible so that it can be optimized for various applications.

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Acknowledgements

We thank V. Surendranath for help with photography, H. Bellen (Baylor College of Medicine, Texas) for the Csp-sGFP transgene, and S. Singh, Sonal and S. Simmert for seeding the wiki with material during the Dresden International PhD Program course. We thank B. Cox and G. Petry of the Morgridge Institute for Research for assistance with 3D printing and parts fabrication. J.S., L.S. and K.W.E. were supported by US National Institutes of Health grants RC2GM092519 and R01CA136590. S.P. was supported by the Human Frontier Science Program Postdoctoral Fellowship. P.T. and P.G.P. were supported by the European Research Council Community's Seventh Framework Programme (FP7/2007-2013) grant agreement 260746.

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Author notes

  1. Peter G Pitrone and Johannes Schindelin: These authors contributed equally to this work.

Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Peter G Pitrone, Stephan Preibisch, Michael Weber, Jan Huisken & Pavel Tomancak
  2. Laboratory for Optical and Computational Instrumentation, University of Wisconsin–Madison, Madison, Wisconsin, USA
    Johannes Schindelin, Luke Stuyvenberg & Kevin W Eliceiri
  3. Albert Einstein College of Medicine, New York, Bronx, USA
    Stephan Preibisch

Authors

  1. Peter G Pitrone
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  2. Johannes Schindelin
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  3. Luke Stuyvenberg
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  4. Stephan Preibisch
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  5. Michael Weber
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  6. Kevin W Eliceiri
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  7. Jan Huisken
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  8. Pavel Tomancak
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Corresponding author

Correspondence toPavel Tomancak.

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The authors declare no competing financial interests.

Supplementary information

Supplementary

Supplementary Figures 1–4 (PDF 6083 kb)

Stitched zebrafish larva

Sweep through the 3D volume of 2-d-old living zebrafish larva mounted in 1% agarose expressing H2A-EGFP under the control of β-actin promoter (Tg(Bactin:H2A-EGFP)) in all cells imaged as a set of six overlapping fields of view of 80 slices 3 μm apart. The stacks were montaged using Fiji's stitching plug-in with the maximum-intensity fusion method. http://openspim.org/File:Supplementary_Video_1.ogv (MOV 1420 kb)

Beating zebrafish heart

The beating heart of a 2-d-old zebrafish larva expressing the cardiac myosin light chain EGFP fusion Tg(cmlc2:EGFP), serving as a myocardium-specific marker, was imaged with single-plane illumination at ten frames per second (the maximum of the Hamamatsu Orca camera). Two full heart beats per second are captured at this rate. On the left side the fluorescence of the heart reporter is captured together with the bright-field image revealing the anatomical context. http://openspim.org/File:Supplementary_Video_2.ogv (MOV 3910 kb)

Deconvolved blastoderm-stage Drosophila embryo

Sweep through the reconstructed volume of a blastoderm-stage Drosophila embryo expressing histone-YFP in all cells imaged from six views at 6-μm steps of the light sheet. The data were reconstructed using Fiji's bead-based registration algorithm and fused using multiview deconvolution (Preibisch et al., unpublished data) for 12 iterations. The left embryo shows the data from the orientation of the first acquired view, the middle embryo shows the same volume after 90° rotation along the anterior-posterior axis (note that data were not acquired along this exact orientation and yet the quality is very similar). The embryo on the right side shows axial resolution of the reconstructed volume approximately orthogonal to the rotation axis. http://openspim.org/File:Supplementary_Video_3.ogv (MOV 10617 kb)

Content-based fused blastoderm–stage Drosophila embryo

Sweep through the reconstructed volume of a blastoderm-stage Drosophila embryo expressing histone-YFP in all cells imaged from six views at 6-μm steps of the light sheet. The data were reconstructed using Fiji's bead-based registration algorithm and fused using the content-based fusion plug-in with blending between views. The left embryo shows the data from the orientation of the first acquired view, the middle embryo shows the same volume after 90° rotation along the anterior-posterior axis (note that data were not acquired along this exact orientation and yet the quality is very similar). The embryo on the right side shows axial resolution of the reconstructed volume roughly orthogonal to the rotation axis. Note the significant increase of the background blur compared to in Supplementary Video 3 showing fusion by multiview deconvolution. http://openspim.org/File:Supplementary_Video_4.ogv (MOV 9621 kb)

3D rendering of Drosophila embyrogenesis

A Drosophila embryo expressing histone-YFP in all cells was imaged from five views every 6 min (acquisition of the five views took 3.5 min at 1-mW laser power, 100-ms exposure time and 50 slices 6 μm apart per view) from gastrulation until hatching. The multiview data were reconstructed using bead-based registration and fused with multiview deconvolution for 15 iterations. A macro script exploiting Fiji's 3D Viewer was used to render the reconstructed volume from each time point from dorsal (top), lateral (middle) and ventral (bottom) viewpoints. Note the residual beads around the sample that come from enhancement of the weak red fluorescent bead signal by the deconvolution procedure. The first 198 time points of 235 time-point time lapse are visualized. http://openspim.org/File:Supplementary_Video_5.ogv (MOV 3947 kb)

Multiview time-lapse of the expression pattern of Csp

A Drosophila embryo expressing the Csp-sGFP protein fusion under native promoter control was imaged from five views every 10 min (acquisition of five views took 4.5 min at 1-mW laser power, 500-ms exposure time and 50 slices 6 μm apart per view). On the left side, maximum-intensity projection along the lateral and dorsal-ventral axis are animated from germband extension stage until late embryogenesis, highlighting the dynamic morphogenetic movement of the nervous system. Csp is expressed in all epidermal cells localized to the membrane, and this signal dominates during earlier time points of the movie. Over time the neuronal signal increases. The blur toward the end of the series is caused by the movement of the living embryo. The maximum-intensity projection on the top right side, roughly alongside the rotation axis, shows that despite the low resolution, the tissue-level expression pattern can be discerned. The bottom right shows a 3D rendering of Csp signal over time using a fixed threshold that isolates the stronger nervous system signal, revealing striking relocation of the brain hemispheres during head involution. http://openspim.org/File:Supplementary_Video_6.ogv (MOV 5072 kb)

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Pitrone, P., Schindelin, J., Stuyvenberg, L. et al. OpenSPIM: an open-access light-sheet microscopy platform.Nat Methods 10, 598–599 (2013). https://doi.org/10.1038/nmeth.2507

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