Fast volumetric calcium imaging across multiple cortical layers using sculpted light

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Abstract

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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Figure 1: Schematic and principle of s-TeFo imaging system.
Figure 2: High-speed single plane Ca2+ imaging in mouse PPC at 158 frames per second (fps) using s-TeFo.
Figure 3: Fast dual-plane Ca2+ imaging in mouse PPC at 10 Hz.
Figure 4: Fast volumetric imaging of Ca2+ dynamics across cortical layers in mouse PPC.
Figure 5: Fast volumetric imaging of Ca2+ dynamics in mouse hippocampus CA1.

Change history

  • 14 November 2016

    In the version of this supplementary file originally posted online, Supplementary Notes 1 and 2 and Supplementary Table 1 were missing. In addition, the link for Supplementary Video 2 directed readers to an unrelated video. The errors have been corrected as of 14 November 2016.

  • 01 December 2016

    In the version of this article initially published online, a grant number was stated incorrectly in the Acknowledgements. The grant number in the sentence "A.F. acknowledges financial support by the European Union (FP7-ICT-217744)" was corrected to FP7-ICT-317744. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

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.

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Authors

Contributions

R.P. contributed to the conceptualization of the imaging approach, designed and built the imaging system, performed experiments, wrote software, analyzed data and wrote the manuscript. A.J.V. designed and built the custom FCPA system together with A.F. under the guidance of A.B. and performed experiments. A.J.P.-A. performed virus injections, cranial window surgeries in hippocampus and cortical areas performed imaging experiments, histology experiments, analyzed data and contributed to the writing of the manuscript. S.W. performed experiments, wrote software, analyzed data and wrote the manuscript. B.S.H. performed the viral injections and cranial window implants for the cortical imaging experiments. B.S.H and P.G. shared expertise on in vivo imaging of awake mice and provided inputs to the final version of the manuscript. T.N. wrote control software and performed in vivo characterization and control experiments, and provided inputs to the final version of the manuscript. J.E.D. wrote analysis code and analyzed data. A.V. conceived and led project, designed imaging system and in vivo mouse experiments, and wrote the manuscript.

Corresponding author

Correspondence to Alipasha Vaziri.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Schematic comparison of TeFo and diffraction-limited excitation.

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.

Supplementary Figure 3 Immunohistochemical assessment of photo-induced damage.

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.

Supplementary Figure 4 Custom animal mount minimizes brain motion during awake imaging.

(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 information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Notes 1 and 2 and Supplementary Table 1 (PDF 1523 kb)

High-speed 2D Ca2+-imaging in mouse posterior parietal cortex.

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)

High-speed 2D Ca2+-imaging in mouse posterior parietal cortex at 470μm depth.

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)

Fast dual-plane Ca2+-imaging in mouse posterior parietal cortex.

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)

High-speed 2D Ca2+-dynamics in mouse hippocampus CA1.

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)

Fast volumetric imaging of 3D Ca2+-dynamics in mouse hippocampus CA1.

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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