Abstract
Spatiotemporal activity patterns in three-dimensionally organized cellular networks are fundamental to the function of the nervous system. Despite advances in functional imaging of cell populations, a method to resolve local network activity in three dimensions has been lacking. Here we introduce a three-dimensional (3D) line-scan technology for two-photon microscopy that permits fast fluorescence measurements from several hundred cells distributed in 3D space. We combined sinusoidal vibration of the microscope objective at 10 Hz with 'smart' movements of galvanometric x-y scanners to repeatedly scan the laser focus along a closed 3D trajectory. More than 90% of cell somata were sampled by the scan line within volumes of 250 μm side length. Using bulk-loading of calcium indicator, we applied this method to reveal spatiotemporal activity patterns in neuronal and astrocytic networks in the rat neocortex in vivo. Two-photon population imaging using 3D scanning opens the field for comprehensive studies of local network dynamics in intact tissue.
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Acknowledgements
We thank J.N.D. Kerr for help with initial experiments, J. Waters for help with LabView programming, H.J. Kasper, J. Tritthardt and S. Giger for excellent technical support, and R.W. Friedrich, J.N.D. Kerr and K.A. Martin for comments on the manuscript. This work was supported by a Human Frontier Science Program research grant to F.H. (RGP0009/2004-C) and a predoctoral fellowship of the Neuroscience Center Zurich to W.G.
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Contributions
W.G. and F.H. devised the method and designed the microscope system; W.G. wrote the software; W.G. and B.M.K. performed the experiments; W.G., B.M.K. and F.H. jointly wrote the manuscript.
Note: Supplementary information is available on the Nature Methods website.
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W.G. and F.H. are authors on a patent application on 3D laser scanning (EP 06 018 315.9).
Supplementary information
Supplementary Fig. 1
Amplitude and phase correction of the z-scan signal.
Supplementary Fig. 2
Schematic illustration of the calculation of a smooth scan trajectory in user-defined mode.
Supplementary Fig. 3
Equidistant distribution of final scan points along the scan trajectory and interpolation of scan segments in user-defined mode.
Supplementary Fig. 4
Stability of 3D scanning over time.
Supplementary Video 1
Live movie of a 3D scan trajectory. A two-photon excited fluorescence spot was imaged using a 10× objective and a video camera at different objective vibration frequencies. 3D spiral scanning is shown in real time first with low than with high zoom. The last frame represents a maximum intensity projection of frames taken at higher magnification.
Supplementary Video 2
3D movie of the neocortical network activity shown in Fig. 5a and b. Calcium dynamics in 205 cells is visualized by assigning fluorescence signals (DF/F) obtained with 3D scanning to the cells using two different color-lookup tables for neurons and astrocytes, respectively (black spheres: neurons at rest; blue spheres: astrocytes at rest; small grey spheres: cells not sampled by the 3D linescan). Electrical stimulation through an extracellular micropipette is indicated (yellow: no stimulation; red: stimulation). Each stimulus is a burst of 10 current pulses delivered at 100 Hz. Note the neuronal responses and the calcium wave in the astrocyte population in layer 1. Speed: 2.5x accelerated.
Supplementary Video 3
3D movie of the neocortical network activity shown in Fig. 5c and d. Calcium dynamics in 375 cells is visualized by assigning fluorescence signals (DF/F) obtained with 3D scanning to the cells using two different color-lookup tables for neurons and astrocytes, respectively (black spheres: neurons at rest; blue spheres: astrocytes at rest; small grey spheres: cells not sampled by the 3D linescan). Electrical stimulation through an extracellular micropipette is indicated (yellow: no stimulation; red: stimulation). Each stimulus is a single current pulse evoking calcium transients mainly close to the pipette tip. Speed: 2.5× accelerated.
Supplementary Video 4
3D movie of spontaneous epileptic-like neocortical network activity after local injection of the GABAA receptor antagonist bicuculline. Highly-synchronized large neuronal and astrocytic calcium transients are visualized as described in the legend to Supplementary Video 3. In addition, the simultaneously recorded local field potential (LFP) is shown at the bottom demonstrating synchrony between the electrical excitation and calcium dynamics. Speed: 2.5x accelerated.
Supplementary Software
ZIP file containing LabView virtual instruments (VIs) of the core routines used for the generation of 3D scan trajectories in the different modes.
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Göbel, W., Kampa, B. & Helmchen, F. Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4, 73–79 (2007). https://doi.org/10.1038/nmeth989
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DOI: https://doi.org/10.1038/nmeth989
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