Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

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.


  1. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    Article  Google Scholar 

  2. Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  ADS  Google Scholar 

  3. Dodt, H.-U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nature Methods 4, 331–336 (2007).

    Article  Google Scholar 

  4. Verveer, P. J. et al. High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nature Methods 4, 311–313 (2007).

    Article  Google Scholar 

  5. Keller, P. J. et al. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nature Methods 7, 637–642 (2010).

    Article  Google Scholar 

  6. Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M. & Keller, P. J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nature Methods 10, 413–420 (2013).

    Article  Google Scholar 

  7. Fahrbach, F. O., Voigt, F. F., Schmid, B., Helmchen, F. & Huisken, J. Rapid 3D light-sheet microscopy with a tunable lens. Opt. Express 21, 21010–21026 (2013).

    Article  ADS  Google Scholar 

  8. Holekamp, T. F., Turaga, D. & Holy, T. E. Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy. Neuron 57, 661–672 (2008).

    Article  Google Scholar 

  9. Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 108, 17708–17713 (2011).

    Article  ADS  Google Scholar 

  10. Kumar, S. et al. High-speed 2D and 3D fluorescence microscopy of cardiac myocytes. Opt. Express 19, 13839–13847 (2011).

    Article  ADS  Google Scholar 

  11. Kumar, S. et al. High-speed 2D and 3D fluorescence microscopy of cardiac myocytes. Opt. Express 19, 13839–13847 (2011).

    Article  ADS  Google Scholar 

  12. Gobel, W., Kampa, B. M. & Helmchen, F. Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nature Methods 4, 73–79 (2007).

    Article  Google Scholar 

  13. Glickfeld, L. L., Andermann, M. L., Bonin, V. & Reid, R. C. Cortico-cortical projections in mouse visual cortex are functionally target specific. Nature Neurosci. 16, 219–226 (2013).

    Article  Google Scholar 

  14. Jia, H., Varga, Z., Sakmann, B. & Konnerth, A. Linear integration of spine Ca2+ signals in layer 4 cortical neurons in vivo. Proc. Natl Acad. Sci. USA 111, 9277–9282 (2014).

    Article  ADS  Google Scholar 

  15. Mittmann, W. et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nature Neurosci. 14,1089–1093 (2011).

    Article  Google Scholar 

  16. Schrodel, T., Prevedel, R., Aumayr, K., Zimmer, M. & Vaziri, A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nature Methods 10, 1013–1020 (2013).

    Article  Google Scholar 

  17. Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nature Methods 9, 201–208 (2012).

    Article  Google Scholar 

  18. Grewe, B. F., Langer, D., Kasper, H., Kampa, B. M. & Helmchen, F. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nature Methods 7, 399–405 (2010).

    Article  Google Scholar 

  19. Cotton, R. J., Froudarakis, E., Storer, P., Saggau, P. & Tolias, A. S. Three-dimensional mapping of microcircuit correlation structure. Front. Neural Circ. 7, 151 (2013).

    Google Scholar 

  20. Dwyer, P. J., DiMarzio, C. A., Zavislan, J. M., Fox, W. J. & Rajadhyaksha, M. Confocal reflectance theta line scanning microscope for imaging human skin in vivo. Opt. Lett. 31, 942–944 (2006).

    Article  ADS  Google Scholar 

  21. Dunsby, C. Optically sectioned imaging by oblique plane microscopy. Opt. Express 16, 20306–20316 (2008).

    Article  ADS  Google Scholar 

  22. Vaziri, A. & Shank, C. V. Ultrafast widefield optical sectioning microscopy by multifocal temporal focusing. Opt. Express 18, 19645–19655 (2010).

    Article  ADS  Google Scholar 

  23. Hillman, E. M. C. & Moore, A. All-optical anatomical co-registration for molecular imaging of small animals using dynamic contrast. Nature Photon. 1, 526–530 (2007).

    Article  ADS  Google Scholar 

  24. Schuster, C. M., Davis, G. W., Fetter, R. D. & Goodman, C. S. Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. Neuron 17, 641–654 (1996).

    Article  Google Scholar 

  25. Curtis, N. J., Ringo, J. M. & Dowse, H. B. Morphology of the pupal heart, adult heart, and associated tissues in the fruit fly, Drosophila melanogaster. J. Morphol. 240, 225–235 (1999).

    Article  Google Scholar 

  26. Bouchard, M. B. et al. Technical considerations in longitudinal multispectral small animal molecular imaging. J. Biomed. Opt. 12, 051601 (2007).

    Article  ADS  MathSciNet  Google Scholar 

  27. Broxton, M. et al. Wave optics theory and 3-D deconvolution for the light field microscope. Opt. Express 21, 25418–25439 (2013).

    Article  ADS  Google Scholar 

  28. Quirin, S., Jackson, J., Peterka, D. S. & Yuste, R. Simultaneous imaging of neural activity in three dimensions. Front. Neural Circ. 8, 29 (2014).

    Google Scholar 

  29. Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013).

    Article  Google Scholar 

  30. Hillman, E. M. C., Boas, D. A., Dale, A. M. & Dunn, A. K. Laminar optical tomography: demonstration of millimeter-scale depth-resolved imaging in turbid media. Opt. Lett. 29, 1650–1652 (2004).

    Article  ADS  Google Scholar 

  31. Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M. & Fraser, S. E. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nature Methods 8, 757–760 (2011).

    Article  Google Scholar 

  32. Horton, N. G. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nature Photon. 7, 205–209 (2013).

    Article  ADS  Google Scholar 

  33. Kobat, D. et al. Deep tissue multiphoton microscopy using longer wavelength excitation. Opt. Express 17, 13354–13364 (2009).

    Article  ADS  Google Scholar 

  34. Lavagnino, Z., Zanacchi, F. C., Ronzitti, E. & Diaspro, A. Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: characterization and application. Opt. Express 21, 5998–6008 (2013).

    Article  ADS  Google Scholar 

  35. Friedrich, M., Gan, Q., Ermolayev, V. & Harms, G. S. STED-SPIM stimulated emission depletion improves sheet illumination microscopy resolution. Biophys. J. 100, L43–L45 (2011).

    Article  Google Scholar 

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

    Article  Google Scholar 

  37. Lutz, C. et al. Holographic photolysis of caged neurotransmitters. Nature Methods 5, 821–827 (2008).

    Article  Google Scholar 

  38. Golan, L., Reutsky, I., Farah, N. & Shoham, S. Design and characteristics of holographic neural photo-stimulation systems. J. Neural Eng. 6, 066004 (2009).

    Article  ADS  Google Scholar 

  39. Jing, D. et al. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. FASEB J. 28, 1582–1592 (2014).

    Article  Google Scholar 

  40. Carlson, G. C. & Coulter, D. A. In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording. Nature Protoc. 3, 249–255 (2008).

    Article  Google Scholar 

  41. Xie, W. et al. Imaging atrial arrhythmic intracellular calcium in intact heart. J. Mol. Cell. Cardiol. 64, 120–123 (2013).

    Article  Google Scholar 

  42. Sung, Y. et al. Three-dimensional holographic refractive-index measurement of continuously flowing cells in a microfluidic channel. Phys. Rev. Appl. 1, 014002 (2014).

    Article  ADS  Google Scholar 

  43. Regmi, R., Mohan, K. & Mondal, P. P. Light sheet based imaging flow cytometry on a microfluidic platform. Microsc. Res. Tech. 76, 1101–1107 (2013).

    Article  Google Scholar 

  44. Baik, A. D., Qiu, J., Hillman, E. M. C., Dong, C. & Guo, X. E. Simultaneous tracking of 3D actin and microtubule strains in individual MLO-Y4 osteocytes under oscillatory flow. Biochem. Biophys. Res. Commun. 431, 718–723 (2013).

    Article  Google Scholar 

  45. Radosevich, A. J., Bouchard, M. B., Burgess, S. A., Chen, B. R. & Hillman, E. M. C. Hyperspectral in vivo two-photon microscopy of intrinsic contrast. Opt. Lett. 33, 2164–2166 (2008).

    Article  ADS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Ethics declarations

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).

Supplementary information

Supplementary information

Supplementary information (PDF 3830 kb)

Supplementary information

Supplementary information (PDF 579 kb)

Supplementary movie 1

Supplementary movie 1 (MOV 29435 kb)

Supplementary movie 2

Supplementary movie 2 (MOV 2447 kb)

Supplementary movie 3

Supplementary movie 3 (MOV 28204 kb)

Supplementary movie 4

Supplementary movie 4 (MOV 19884 kb)

Supplementary movie 5

Supplementary movie 5 (MOV 12484 kb)

Supplementary movie 6

Supplementary movie 6 (MOV 7745 kb)

Supplementary movie 7

Supplementary movie 7 (MOV 16905 kb)

Supplementary movie 8

Supplementary movie 8 (MOV 13566 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing