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Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain


Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

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All custom code generated for this study can be obtained from the corresponding authors on reasonable request.

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The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information. All datasets generated during this study are available from the corresponding authors.

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  1. 1.

    Hilgetag, C. C. & Amunts, K. Connectivity and cortical architecture. eNeuroforum 7, 56–63 (2016).

  2. 2.

    Peron, S., Chen, T. W. & Svoboda, K. Comprehensive imaging of cortical networks. Curr. Opin. Neurobiol. 32, 115–123 (2015).

  3. 3.

    Eggebrecht, A. T. et al. Mapping distributed brain function and networks with diffuse optical tomography. Nat. Photon. 8, 448–454 (2014).

  4. 4.

    Errico, C. et al. Transcranial functional ultrasound imaging of the brain using microbubble-enhanced ultrasensitive Doppler. NeuroImage 124, 752–761 (2016).

  5. 5.

    Schulz, K. et al. Simultaneous BOLD fMRI and fiber-optic calcium recording in rat neocortex. Nat. Methods 9, 597–602 (2012).

  6. 6.

    Looger, L. L. & Griesbeck, O. Genetically encoded neural activity indicators. Curr. Opin. Neurobiol. 22, 18–23 (2012).

  7. 7.

    Yang, W. & Yuste, R. In vivo imaging of neural activity. Nat. Methods 14, 349–359 (2017).

  8. 8.

    Bouchard, M. B. et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Nat. Photon. 9, 113–119 (2015).

  9. 9.

    Dana, H. et al. Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo. PLoS ONE 9, e108697 (2014).

  10. 10.

    Prevedel, R. et al. Fast volumetric calcium imaging across multiple cortical layers using sculpted light. Nat. Methods 13, 1021–1028 (2016).

  11. 11.

    Dean-Ben, X. L. et al. Functional optoacoustic neuro-tomography for scalable whole-brain monitoring of calcium indicators. Light Sci. Appl. 5, e16201 (2016).

  12. 12.

    Gottschalk, S., Fehm, T. F., Dean-Ben, X. L., Tsytsarev, V. & Razansky, D. Correlation between volumetric oxygenation responses and electrophysiology identifies deep thalamocortical activity during epileptic seizures. Neurophotonics 4, 011007 (2017).

  13. 13.

    Tang, J., Coleman, J. E., Dai, X. & Jiang, H. Wearable 3-D photoacoustic tomography for functional brain imaging in behaving rats. Sci. Rep. 6, 25470 (2016).

  14. 14.

    Dean-Ben, X. L., Gottschalk, S., Sela, G., Shoham, S. & Razansky, D. Functional optoacoustic neuro-tomography of calcium fluxes in adult zebrafish brain in vivo. Opt. Lett. 42, 959–962 (2017).

  15. 15.

    Wang, L. V. & Yao, J. A practical guide to photoacoustic tomography in the life sciences. Nat. Methods 13, 627–638 (2016).

  16. 16.

    Ermolayev, V., Dean-Ben, X. L., Mandal, S., Ntziachristos, V. & Razansky, D. Simultaneous visualization of tumour oxygenation, neovascularization and contrast agent perfusion by real-time three-dimensional optoacoustic tomography. Eur. Radiol. 26, 1843–1851 (2016).

  17. 17.

    Gottschalk, S., Fehm, T. F., Dean-Ben, X. L. & Razansky, D. Noninvasive real-time visualization of multiple cerebral hemodynamic parameters in whole mouse brains using five-dimensional optoacoustic tomography. J. Cereb. Blood Flow Metab. 35, 531–535 (2015).

  18. 18.

    Knieling, F. et al. Multispectral optoacoustic tomography for assessment of Crohn’s disease activity. New Engl. J. Med. 376, 1292–1294 (2017).

  19. 19.

    Tzoumas, S. et al. Eigenspectra optoacoustic tomography achieves quantitative blood oxygenation imaging deep in tissues. Nat. Commun. 7, 12121 (2016).

  20. 20.

    Yao, J. et al. High-speed label-free functional photoacoustic microscopy of mouse brain in action. Nat. Methods 12, 407–410 (2015).

  21. 21.

    Schmued, L., Kyriakidis, K. & Heimer, L. In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextran-amine, Fluoro-Ruby, within the CNS. Brain Res. 526, 127–134 (1990).

  22. 22.

    Bojak, I., Day, H. C. & Liley, D. T. Ketamine, propofol, and the EEG: a neural field analysis of HCN1-mediated interactions. Front. Comput. Neurosci. 7, 22 (2013).

  23. 23.

    Dhir, A. Pentylenetetrazol (PTZ) kindling model of epilepsy.Curr. Protoc. Neurosci. 58, 9.37.1–9.37.12 (2012).

  24. 24.

    Tang, J. et al. Noninvasive high-speed photoacoustic tomography of cerebral hemodynamics in awake-moving rats. J. Cereb. Blood Flow Metab. 35, 1224–1232 (2015).

  25. 25.

    Durán-Riveroll, M. L. & Cembella, D. A. Guanidinium toxins and their interactions with voltage-gated sodium ion channels. Mar. Drugs 15, 303 (2017).

  26. 26.

    Gottschalk, S. et al. Short and long-term phototoxicity in cells expressing genetic reporters under nanosecond laser exposure. Biomaterials 69, 38–44 (2015).

  27. 27.

    Norup Nielsen, A. & Lauritzen, M. Coupling and uncoupling of activity-dependent increases of neuronal activity and blood flow in rat somatosensory cortex. J. Physiol. 533, 773–785 (2001).

  28. 28.

    Kozberg, M. G., Ma, Y., Shaik, M. A., Kim, S. H. & Hillman, E. M. Rapid postnatal expansion of neural networks occurs in an environment of altered neurovascular and neurometabolic coupling. J. Neurosci. 36, 6704–6717 (2016).

  29. 29.

    Vanni, M. P. & Murphy, T. H. Mesoscale transcranial spontaneous activity mapping in GCaMP3 transgenic mice reveals extensive reciprocal connections between areas of somatomotor cortex. J. Neurosci. 34, 15931–15946 (2014).

  30. 30.

    Schroeter, A., Grandjean, J., Schlegel, F., Saab, B. J. & Rudin, M. Contributions of structural connectivity and cerebrovascular parameters to functional magnetic resonance imaging signals in mice at rest and during sensory paw stimulation. J. Cereb. Blood Flow Metab. 37, 2368–2382 (2017).

  31. 31.

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

  32. 32.

    O’Herron, P. et al. Neural correlates of single-vessel haemodynamic responses in vivo. Nature 534, 378–382 (2016).

  33. 33.

    Razi, A. & Friston, K. J. The connected brain: causality, models, and intrinsic dynamics. IEEE Signal Process. Mag. 33, 14–35 (2016).

  34. 34.

    Lefebvre, J., Castonguay, A., Pouliot, P., Descoteaux, M. & Lesage, F. Whole mouse brain imaging using optical coherence tomography: reconstruction, normalization, segmentation, and comparison with diffusion MRI. Neurophotonics 4, 041501 (2017).

  35. 35.

    Llinás, R. R., Leznik, E. & Urbano, F. J. Temporal binding via cortical coincidence detection of specific and nonspecific thalamocortical inputs: a voltage-dependent dye-imaging study in mouse brain slices. Proc. Natl Acad. Sci. USA 99, 449–454 (2002).

  36. 36.

    Kneipp, M. et al. Effects of the murine skull in optoacoustic brain microscopy. J. Biophotonics 9, 117–123 (2016).

  37. 37.

    Sieu, L. A. et al. EEG and functional ultrasound imaging in mobile rats. Nat. Methods 12, 831–834 (2015).

  38. 38.

    Badura, A., Sun, X. R., Giovannucci, A., Lynch, L. A. & Wang, S. S. Fast calcium sensor proteins for monitoring neural activity. Neurophotonics 1, 025008 (2014).

  39. 39.

    Dana, H. et al. High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments. Preprint at (2018).

  40. 40.

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

  41. 41.

    Qian, Y. et al. A genetically encoded near-infrared fluorescent calcium ion indicator.Nat. Methods 16, 171–174 (2019).

  42. 42.

    American National Standard for Safe Use of Lasers ANSI Z136.1 (Laser Institute of America, 2014).

  43. 43.

    Dean-Ben, X. L., Ozbek, A. & Razansky, D. Volumetric real-time tracking of peripheral human vasculature with GPU-accelerated three-dimensional optoacoustic tomography. IEEE Trans. Med. Imaging 32, 2050–2055 (2013).

  44. 44.

    Wang, L. V. & Wu, H.-I. Biomedical Optics: Principles and Imaging (Wiley, 2007).

  45. 45.

    Abramowitz, M. & Stegun, I. A. Handbook of Mathematical Functions: With Formulas, Graphs, and Mathematical Tables Vol. 55 (Courer Corporation, 1964).

  46. 46.

    Zarchan, P. & Musoff, H. Fundamentals of Kalman Filtering: A Practical Approach (American Institute of Aeronautics and Astronautics, 2000).

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The authors acknowledge grant support from the European Research Council (under grant agreement ERC-2015-CoG-682379) and the US National Institutes of Health (grants R21-EY026382 and UF1-NS107680). We also acknowledge the help of N. Tritsch and L. Mcley with reading and commenting on the manuscript.

Author information

S.G., O.D., B.M.L., M.A.H. and X.L.D.-B. performed the experiments. S.G., O.D., B.M.L., J.R., M.A.H. and X.L.D.-B. analysed and processed the data. S.G., X.L.D.-B., S.S. and D.R. validated the data analysis. S.G., S.S. and D.R. designed and supervised the study. All authors contributed to writing the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Daniel Razansky.

Supplementary information

  1. Supplementary Information

    Supplementary figures and video captions.

  2. Reporting Summary

  3. Supplementary Video 1

    OA calcium activity map in a single 2D slice located at an approximate depth of 1 mm in the mouse brain.

  4. Supplementary Video 2

    OA calcium activity in a single 2D slice located at an approximate depth of 0.5 mm in the mouse brain.

  5. Supplementary Video 3

    Haemodynamic responses across the entire mouse cortex in response to paw stimulation.

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Fig. 1: Bi-modal OA and fluorescence imaging of isolated brains.
Fig. 2: Whole-brain volumetric OA imaging of neuronal activation in the isolated brain model.
Fig. 3: Non-invasive imaging of the GCaMP6f brain in vivo.
Fig. 4: Non-invasive imaging of somatosensory-evoked rapid calcium transients in the GCaMP6f brain in vivo.
Fig. 5: Comparison of GCaMP6f and GCaMP6s responses to electrical stimulation of the right or left hind paw.