Although whole-organism calcium imaging in small and semi-transparent animals has been demonstrated, capturing the functional dynamics of large-scale neuronal circuits in awake behaving mammals at high speed and resolution has remained one of the main frontiers in systems neuroscience. Here we present a method based on light sculpting that enables unbiased single- and dual-plane high-speed (up to 160 Hz) calcium imaging as well as in vivo volumetric calcium imaging of a mouse cortical column (0.5 mm × 0.5 mm × 0.5 mm) at single-cell resolution and fast volume rates (3–6 Hz). We achieved this by tailoring the point-spread function of our microscope to the structures of interest while maximizing the signal-to-noise ratio using a home-built fiber laser amplifier with pulses that are synchronized to the imaging voxel speed. This enabled in vivo recording of calcium dynamics of several thousand neurons across cortical layers and in the hippocampus of awake behaving mice.
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Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).
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. Nat. Methods 10, 413–420 (2013).
Chen, B.-C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).
Levoy, M., Ng, R., Adams, A., Footer, M. & Horowitz, M. Light field microscopy. ACM Trans. Graph. 25, 924–934 (2006).
Prevedel, R. et al. Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nat. Methods 11, 727–730 (2014).
Bouchard, M.B. et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Nat. Photonics 9, 113–119 (2015).
Ahrens, M.B. et al. Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nature 485, 471–477 (2012).
Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).
Hoover, E.E. & Squier, J.A. Advances in multiphoton microscopy technology. Nat. Photonics 7, 93–101 (2013).
Mittmann, W. et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nat. Neurosci. 14, 1089–1093 (2011).
Thériault, G., Cottet, M., Castonguay, A., McCarthy, N. & De Koninck, Y. Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging. Front. Cell. Neurosci. 8, 139 (2014).
Botcherby, E.J. et al. Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. Proc. Natl. Acad. Sci. USA 109, 2919–2924 (2012).
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. Nat. Methods 7, 399–405 (2010).
Kirkby, P.A., Srinivas Nadella, K.M.N. & Silver, R.A. A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy. Opt. Express 18, 13721–13745 (2010).
Kong, L. et al. Continuous volumetric imaging via an optical phase-locked ultrasound lens. Nat. Methods 12, 759–762 (2015).
Duemani Reddy, G., Kelleher, K., Fink, R. & Saggau, P. Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat. Neurosci. 11, 713–720 (2008).
Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201–208 (2012).
Fernández-Alfonso, T. et al. Monitoring synaptic and neuronal activity in 3D with synthetic and genetic indicators using a compact acousto-optic lens two-photon microscope. J. Neurosci. Methods 222, 69–81 (2014).
Kim, K.H. et al. Multifocal multiphoton microscopy based on multianode photomultiplier tubes. Opt. Express 15, 11658–11678 (2007).
Stirman, J.N., Smith, I.T., Kudenov, M.W. & Smith, S.L. Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain. Nat. Biotechnol. 34, 857–862 (2016).
Cheng, A., Gonçalves, J.T., Golshani, P., Arisaka, K. & Portera-Cailliau, C. Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat. Methods 8, 139–142 (2011).
Yang, S.J. et al. Extended field-of-view and increased-signal 3D holographic illumination with time-division multiplexing. Opt. Express 23, 32573–32581 (2015).
Yang, W. et al. Simultaneous Multi-plane Imaging of Neural Circuits. Neuron 89, 269–284 (2016).
Mountcastle, V.B. The columnar organization of the neocortex. Brain 120, 701–722 (1997).
Oron, D., Tal, E. & Silberberg, Y. Scanningless depth-resolved microscopy. Opt. Express 13, 1468–1476 (2005).
Zhu, G., van Howe, J., Durst, M., Zipfel, W. & Xu, C. Simultaneous spatial and temporal focusing of femtosecond pulses. Opt. Express 13, 2153–2159 (2005).
Schrödel, T., Prevedel, R., Aumayr, K., Zimmer, M. & Vaziri, A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat. Methods 10, 1013–1020 (2013).
Dana, H. et al. Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks. Nat. Commun. 5, 3997 (2014).
Andrasfalvy, B.K., Zemelman, B.V., Tang, J. & Vaziri, A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl. Acad. Sci. U.S.A. 107, 11981–11986 (2010).
Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).
Rickgauer, J.P., Deisseroth, K. & Tank, D.W. Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields. Nat. Neurosci. 17, 1816–1824 (2014).
Therrien, O.D., Aubé, B., Pagès, S., Koninck, P.D. & Côté, D. Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination. Biomed. Opt. Express 2, 696–704 (2011).
Choi, H. et al. 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation. Opt. Express 20, 26219–26235 (2012).
Kandel, E.R., Schwartz, J.H. & Jessell, T.M. Principles of Neural Science (McGraw-Hill, Health Professions Division, 2000).
Kalmbach, A.S. & Waters, J. Brain surface temperature under a craniotomy. J. Neurophysiol. 108, 3138–3146 (2012).
Stujenske, J.M., Spellman, T. & Gordon, J.A. Modeling the spatiotemporal dynamics of light and heat propagation for in vivo optogenetics. Cell Rep. 12, 525–534 (2015).
Podgorski, K. & Ranganathan, G. Brain heating induced by near-infrared lasers during multiphoton microscopy. J. Neurophysiol. 116, 1012–1023 (2016).
Koester, H.J., Baur, D., Uhl, R. & Hell, S.W. Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236 (1999).
Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).
Mukamel, E.A., Nimmerjahn, A. & Schnitzer, M.J. Automated analysis of cellular signals from large-scale calcium imaging data. Neuron 63, 747–760 (2009).
Pnevmatikakis, E.A. et al. Simultaneous denoising, deconvolution, and demixing of calcium imaging data. Neuron 89, 285–299 (2016).
Atlas, A.M.B. Allen Mouse Brain Atlas [Internet]., Available from: http://mouse.brain-map.org (2015).
Dombeck, D.A., Harvey, C.D., Tian, L., Looger, L.L. & Tank, D.W. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat. Neurosci. 13, 1433–1440 (2010).
Kaifosh, P., Lovett-Barron, M., Turi, G.F., Reardon, T.R. & Losonczy, A. Septo-hippocampal GABAergic signaling across multiple modalities in awake mice. Nat. Neurosci. 16, 1182–1184 (2013).
Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).
Rao, R.P.N. & Ballard, D.H. Predictive coding in the visual cortex: a functional interpretation of some extra-classical receptive-field effects. Nat. Neurosci. 2, 79–87 (1999).
Buonomano, D.V. & Maass, W. State-dependent computations: spatiotemporal processing in cortical networks. Nat. Rev. Neurosci. 10, 113–125 (2009).
Sompolinsky, H. Computational neuroscience: beyond the local circuit. Curr. Opin. Neurobiol. 25, xiii–xviii (2014).
Bewersdorf, J., Pick, R. & Hell, S.W. Multifocal multiphoton microscopy. Opt. Lett. 23, 655–657 (1998).
Pologruto, T.A., Sabatini, B.L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).
Chong, A., Renninger, W.H. & Wise, F.W. Properties of normal-dispersion femtosecond fiber lasers. J. Opt. Soc. Am. B 25, 140–148 (2008).
Zhu, L. et al. Generation of high fidelity 62-fs, 7-nJ pulses at 1035 nm from a net normal-dispersion Yb-fiber laser with anomalous dispersion higher-order-mode fiber. Opt. Express 21, 16255–16262 (2013).
Grüner-Nielsen, L., Jakobsen, D., Jespersen, K.G. & Pálsdóttir, B. A stretcher fiber for use in fs chirped pulse Yb amplifiers. Opt. Express 18, 3768–3773 (2010).
Fernández, A. et al. High-fidelity, 160 fs, 5 μJ pulses from an integrated Yb-fiber laser system with a fiber stretcher matching a simple grating compressor. Opt. Lett. 37, 927–929 (2012).
We thank W. Haubensak (Institute for Molecular Pathology (IMP), Vienna) and their lab members for sharing the satellite mouse facility and reagents and S. Rumpel and D. Aschauer (IMP) for providing mice during the initial tests, A. Piszczek and G. Petri for immunohistochemistry and slice imaging, G. Keller (Friedrich Miescher Institute, Basel) for sharing of red calcium indicators, L. Grüner-Nielsen for providing and optimizing the dispersion compensating fiber stretcher module, M. Colombini and the IMP workshop for manufacturing of mechanical components, P. Rupprecht for helpful discussions, G. Jaindl for technical support, and O. Olsen for reading and feedback on the manuscript. R.P. acknowledges the Vienna International Postdoctoral Program (VIPS) Program of the Austrian Federal Ministry of Science and Research and the City of Vienna and the European Commission (Marie Curie, FP7-PEOPLE-2011-IIF). A.F. acknowledges financial support by the European Union (FP7-ICT-317744). This work was supported through funding from the Vienna Science and Technology Fund (WWTF) project VRG10-11, the Human Frontiers Science Program Project RGP0041/2012, the Research Platform Quantum Phenomena and Nanoscale Biological Systems (QuNaBioS), the Institute of Molecular Pathology, the US National Institutes of Health (NIH) award 1U01NS094263-01, the Intelligence Advanced Research Projects Activity (IARPA) via Department of Interior/Interior Business Center (DoI/IBC) contract number D16PC00002. The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright annotation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of IARPA, DoI/IBC, or the US Government.
The authors declare no competing financial interests.
Integrated supplementary information
In conventional two-photon imaging, a neuronal cell body is excited by a set of small (diffraction-limited) PSFs, whose signals are integrated (left). In contrast, in scanned TeFo, the whole neuron is sampled with a single, enlarged sculpted PSF (right). Here V is the (volume) ratio of the diffraction limited spot compared to the enlarged, sculpted TeFo spot.
Supplementary Figure 2 Home-built ytterbium (Yb) all-fiber chirped-pulse laser amplifier (FCPA) design and characterization.
(a) Detailed sketch of the FCPA, showing the Yb-fiber seed oscillator (pulse repetition rate 58 MHz) at the top. LD – laser diode; PC – polarization controller; DCF – dispersion compensating fiber; FCAOM – fiber-coupled acousto-optic modulator; PLMA – polarization-maintaining large mode area fiber; PLMA10 – 10 μm core diameter PLMA; PLMA30 – 30 μm core diameter PLMA. The oscillator is pumped with a 600 mW single-mode 976-nm laser diode. The amplifier is pumped with 10W ~980-nm pump diodes. (b) Left: Output spectral intensity (black) as well as phase (red) of the FCPA; Right: Temporal pulse profile (black - measured FWHM 183 fs, blue - calculated Fourier limited pulse duration based on the output spectrum, FWHM 170fs) and temporal phase profile (red). (c) Measured beam profile of the FCPA, showing corresponding cross sections (white). Scale bar 1mm.
Representative images of brain sections containing the laser scanned regions that were immunostained with antibodies to detect microglial (anti-Iba1; green) and astrocyte (anti-GFAP; red) activation. All brains (with the exception of control) were illuminated for continuous 20min under the condition stated below. Arrows indicate the approximate center of imaging site. (a) Immunolabeled sections of a mouse brain imaged with 2D s-TeFo scanning at 350μm depth with 220 mW effective average laser power. Condition is similar to Fig. 2e. (b) Same as in a, but with 3D s-Tefo from 0-500μm depth and 70-200mW cycled averaged laser power. Condition is the same as in Fig. 4. (c) Control group that underwent window surgery but was not exposed to laser illumination. (d) Positive control group that was subject to 2D s-TeFo at 200μm depth and high laser power (400mW). (e,f) Intensity of immunolabeling, as fraction compared to control area, for mice illuminated with different laser intensities and scanning modalities, as indicated. 2P – standard two-photon microscopy. Shaded area denotes the 95% confidence interval of the control group mean.
(a) Schematics of the custom animal mount utilized in the experiments. The mouse is head restrained but can freely move on a rotating disk. The disk is suspended by springs and a damped counter weight and the animal is held up by a custom jacket (see Fig. 2b). These measures reduce and compensate for any force that is applied by the mouse’s limbs and thus minimize the vertical motion of the mouse brain during active behavior (b) Typical measured lateral drift of the images during the experiment before (blue) and after (green) motion correction in image postprocessing. In general, motion is <5μm r.m.s before and <1μm after correction. Dataset is the same as in Fig. 2c, motion correction algorithm is based on tracking the peak of the image autocorrelation using a maximum likelihood estimation algorithm and subsequent sub-pixel shifting using image interpolation. The high frame rate of s-TeFo (160 Hz) further facilitated image motion correction, as the in-frame movement during the acquisition of a single frame becomes negligible. (c,d) Representative calcium traces extracted before (blue) and after (green) motion correction in image postprocessing. Note that the curves are deliberately offset vertically from each other. (e) Histogram of correlations between corrected and uncorrected traces. On average the correlations are high (R=0.94±0.05). Frame rate is ~160Hz.
Supplementary Figure 5 Comparison of different neuron segmentation and calcium dynamic extraction approaches on s-TeFo imaging data.
ROI maps (left) and activity heat maps (right) for data analysis using PCA/ICA in (a), non-negative matrix factorization (NMF) in (b), and standard-deviation projection in (c). (d) Direct comparison of NMF vs. PCA/ICA analysis. Overlay of example activity traces extracted with PCA/ICA and NMF respectively. (e) Correlation coefficient matrix (left) and correlation coefficient histogram (right) of all neurons detected by both methods. On average the correlations are high (R=0.90±0.04). Comparison was done on data set shown in Figure 2c.
Supplementary Figures 1–5, Supplementary Notes 1 and 2 and Supplementary Table 1 (PDF 1523 kb)
Raw image data of calcium imaging of spontaneous activity of neurons expressing GCaMP6m in mouse posterior parietal cortex. Shown are ~315 seconds of recording at 158Hz acquisition frame rate, with a field-of-view of 500x500μm. Video playback is 5000 frames per second, which is equal to 10 seconds in real time in the video (i.e. playback speed ~30x). To reduce file size frames were binned in groups of 50. Corresponding video to Figure 2c. (MOV 2006 kb)
Raw image data of calcium imaging of spontaneous activity of neurons expressing GCaMP6f in mouse posterior parietal cortex. Shown are ~190 seconds of recording at 158Hz acquisition frame rate, with a field-of-view of 500x500μm at a depth of 470μm from the dura. Video playback is 3000 frames per second, which is equal to 10 seconds in real time in the video (i.e. playback speed ~20x). To reduce file size frames were binned in groups of 50. Corresponding video to Figure 2d. (MOV 1823 kb)
Raw image data of calcium imaging of spontaneous activity of neurons expressing GCaMP6m in mouse posterior parietal cortex. The planes (field-of-view of 500x500μm) were acquired with 10Hz each, in Layer 2/3 at depth of 150μm (left) and Layer 4 at 350μm depth (right) respectively. Shown are ~100 seconds of recording. Video playback is 100 frames per second, which is equal to 10 seconds in real time in the video (i.e. playback speed 10x). Corresponding video to Figure 3b-d. (MOV 1999 kb)
Fast volumetric imaging of 3D Ca2+-dynamics across cortical layers in mouse posterior parietal cortex.
Three-dimensional rendering of raw image data of calcium imaging spontaneous activity of neurons expressing GCaMP6m in mouse posterior parietal cortex. The field-of-view was 500x500x500μm, composed of 43 axial planes, with a volume acquisition rate of 3Hz. Shown are ~330 seconds of recording. Video playback is 75 volumes per second, which is equal to ~13 seconds in real time in the video (i.e. playback speed ~25x). Corresponding video to Figure 4a,d. (MOV 12949 kb)
Raw image data of calcium imaging of spontaneous activity of neurons expressing jRGECKO1a in mouse hippocampus CA1 (overlying cortex aspirated). Shown are ~190 seconds of recording at 158Hz acquisition frame rate, with a field-of-view of 500x500μm. Video playback is 1500 frames per second, which is equal to 20 seconds in real time in the video (i.e. playback speed ~10x). To reduce file size frames were binned in groups of 30. Corresponding video to Figure 5b,c. (MOV 2728 kb)
Three-dimensional rendering of raw image data of calcium imaging spontaneous activity of neurons expressing jRGECKO1a in mouse hippocampus CA1 (overlying cortex aspirated). The field-of-view was 500x500x200μm, composed of 21 axial planes, with a volume acquisition rate of 5.7Hz. Shown are ~265 seconds of recording. Video playback is 60 volumes per second, which is equal to 25 seconds in real time in the video (i.e. playback speed ~10x). Corresponding video to Figure 5d,e. (MOV 10685 kb)
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Prevedel, R., Verhoef, A., Pernía-Andrade, A. et al. Fast volumetric calcium imaging across multiple cortical layers using sculpted light. Nat Methods 13, 1021–1028 (2016). https://doi.org/10.1038/nmeth.4040
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