High-speed, large-scale three-dimensional (3D) imaging of neuronal activity poses a major challenge in neuroscience. Here we demonstrate simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain. Our technique captures the dynamics of spiking neurons in volumes of ∼700 μm × 700 μm × 200 μm at 20 Hz. Its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.
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We thank T. Müller, P. Pasierbek, P. Forai, H. Kaplan, M. Molodtsov, K. Tessmar-Raible, F. Schlumm and Olympus Inc. for technical support and loan of equipment, as well as H. Baier (Max Planck Institute of Neurobiology) and M. Orger (Champalimaud) for sharing zebrafish lines. We thank L. Page for providing early funding for the project and D. Dalrymple for helping catalyze connections. The computational results presented have been achieved in part using the Vienna Scientific Cluster (VSC). This work was supported by the VIPS Program of the Austrian Federal Ministry of Science and Research and the City of Vienna as well as the European Commission (Marie Curie, FP7-PEOPLE-2011-IIF) (R.P.); a Samsung Scholarship (Y.-G.Y.); a US National Science Foundation (NSF) Graduate Fellowship (N.P.); the Allen Institute for Brain Science, the MIT Media Lab, the MIT McGovern Institute, US National Institutes of Health (NIH) 1R01EY023173, the MIT Synthetic Intelligence Project, the Institution of Engineering and Technology (IET) Harvey Prize, NSF CBET 1053233, the New York Stem Cell Foundation–Robertson Award, NSF CBET 1344219, NIH 1DP1NS087724, Google, the NSF Center for Brains, Minds and Machines at MIT, and Jeremy and Joyce Wertheimer (E.S.B.); the Vienna Science and Technology Fund (WWTF) project VRG10-11, Human Frontiers Science Program Project RGP0041/2012, Research Platform Quantum Phenomena and Nanoscale Biological Systems (QuNaBioS) (A.V.); and the European Community's Seventh Framework Programme/ERC no. 281869 (M.Z. and T.S.). The Institute of Molecular Pathology is funded by Boehringer Ingelheim.
The authors declare no competing financial interests.
Supplementary Figures 1–4 and Supplementary Notes 1 and 2 (PDF 6322 kb)
Software for 3D volume reconstruction from light-field images The software for 3D volume reconstruction from light-field images is available as Supplementary Software. It is written in MATLAB and requires a workstation with MATLAB2013a or later version and at least 32GB of RAM. A user manual for the software (LFM_Software_readme.pdf) is included in the .zip file. (ZIP 7047 kb)
Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate (1000 frames in total). Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2a-c. (AVI 7320 kb)
Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2d. (AVI 9412 kb)
Maximum intensity projection of 12 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 15 seconds of recording of basal activity of freely-moving worms at 50 Hz volume rate. Exposure time for individual volumes was 20 ms. Video frame rate is 50 frames per second, which equates to 50 volumes per second in the video (i.e. playback speed 1x – real time). (AVI 9533 kb)
Maximum intensity projection of 15 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity in the head ganglia at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2e-f. (AVI 9673 kb)
Maximum intensity projection of 7 z-planes at 2 μm distance of a of a Punc-31::NLS-GCaMP5K worm during chemosensory stimulation. Shown are 240 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 50 frames per second, which equates to 24 seconds in real time in the video (i.e. playback speed 10x). Also see Supplementary Fig. 3. (AVI 11061 kb)
Maximum intensity projection of 51 z-planes at 4 μm distance of a 5 dpf - HuC:GCaMP5G zebrafish larvae. Shown are 240 seconds of recording of activity at 20 Hz volume rate. Playback speed of video is 10x. At ∼15 sec (∼1.5 sec in the video), decomposed fish water is supplied manually to the fish chamber in order to evoke activity in the olfactory system. Also see Fig. 3 in the main manuscript. (MP4 3748 kb)
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Prevedel, R., Yoon, YG., Hoffmann, M. et al. Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nat Methods 11, 727–730 (2014). https://doi.org/10.1038/nmeth.2964
Imaging volumetric dynamics at high speed in mouse and zebrafish brain with confocal light field microscopy
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