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Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms

Nature Photonics volume 9, pages 113–119 (2015)Cite this article

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Abstract

We report a three-dimensional microscopy technique—swept, confocally-aligned planar excitation (SCAPE) microscopy—that allows volumetric imaging of living samples at ultrahigh speeds. Although confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image three-dimensional volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light sheet in a single-objective, en face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless three-dimensional imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

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Figure 1: SCAPE imaging geometry and image formation.
Figure 2: SCAPE microscopy in mouse brain.
Figure 3: SCAPE microscopy of neuronal calcium dynamics in an awake mouse brain. a,
Figure 4: SCAPE of freely moving mhc-Gal4,UAS-CD8:GFP first instar Drosophila melanogaster larvae.
Figure 5: SCAPE microscopy of cellular structure–function and three-dimensional cell tracking in freely moving Drosophila larvae.

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Acknowledgements

The authors acknowledge the contributions of R. Yuste, D. Kelley and M. Chalfie and their students and staff (in particular M. Agetsuma, S. Quirin and D. Peterka) for assistance with early sample selection and preparation, as well as the Bloomington Stock Center, S. Galindo, M. Baylies and B. Noro for fly stocks. K. Yeager, L.E. Grosberg, M. Shaik, M. Kozberg, S. Kim, Y. Ma, T.J. Muldoon and S. Qian provided assistance with instrumentation and sample preparation. The authors thank I. Herman and T. Galwaduge for assistance with PSF calculations, L. Paninski for discussions on neuronal data analysis and R. Levenson for guidance on applications. The authors acknowledge Lincoln Laser and Cambridge Technology for assistance with scanner fabrication. Funding was provided by NIH (NINDS) R21NS053684, R01 NS076628 and R01NS063226, NSF CAREER 0954796, The Human Frontier Science Program and the Wallace H. Coulter Foundation (E.M.C.H.), NIH (NINDS) R01 NS069679 and the Dana Foundation (R.M.B.), (NINDS) R01NS070644 (R.S.M.), (NINDS) R01NS061908 (W.B.G.), DoD MURI W911NF-12–1-0594 (Yuste). M.B. received NSF and NDSEG graduate fellowships. V.V. was funded by an NSF IGERT Fellowship. C.S.M. is supported by a postdoctoral fellowship from Fundação para a Ciência e a Tecnologia, Portugal.

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Authors and Affiliations

  1. Departments of Biomedical Engineering and Radiology, Laboratory for Functional Optical Imaging, Columbia University, New York, 10027, New York, USA

    Matthew B. Bouchard, Venkatakaushik Voleti & Elizabeth M. C. Hillman

  2. Department of Biochemistry and Molecular Biophysics, Mann Lab, Columbia University, New York, 10032, New York, USA

    César S. Mendes & Richard S. Mann

  3. Department of Neuroscience, Bruno Lab, Columbia University, New York, 10032, New York, USA

    Clay Lacefield & Randy M. Bruno

  4. Department of Physiology and Cellular Biophysics, Department of Neuroscience, College of Physicians and Surgeons, Columbia University Medical Center, New York, 10032, New York, USA

    Wesley B. Grueber

  5. Kavli Institute for Brain Science, Columbia University, New York, New York 10032, USA

    Randy M. Bruno & Elizabeth M. C. Hillman

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Contributions

M.B.B. and E.M.C.H. conceived the technique. M.B.B., E.M.C.H. and V.V. generated ray-tracing models, built the system, acquired and processed data, and prepared the manuscript. C.S.M., R.S.M. and W.B.G. provided and assisted with Drosophila samples, and C.L. and R.M.B. provided and assisted with mouse models. R.S.M., C.S.M., W.B.G., C.L. and R.M.B. all advised on image interpretation and manuscript preparation.

Corresponding author

Correspondence to Elizabeth M. C. Hillman.

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Competing interests

A patent related to this technique was issued to The Trustees of Columbia University in the City of New York on 31 December 2013 (inventors Hillman and Bouchard).

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Bouchard, M., Voleti, V., Mendes, C. et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms. Nature Photon 9, 113–119 (2015). https://doi.org/10.1038/nphoton.2014.323

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