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Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes


The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation–evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.

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Figure 1: Design and characterization of the two-photon microscope setup.
Figure 2: Three-dimensional measurement of BAPs with sub-millisecond resolution.
Figure 3: Point-by-point and continuous 3D trajectory scanning of dendritic Ca2+ spike propagation in CA1 pyramidal cells.
Figure 4: High-speed 3D calcium imaging of spontaneous neuronal network activity in vivo.
Figure 5: 3D measurement of neuronal network activity in vivo in response to visual stimuli.

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We thank A. Csákányi for technical assistance and I. Vanzetta for advice. We thank J. Rátai and D. Rátai for support with the Lenar3Do virtual reality hardware and L. Molnár and G. Karsai for preparing insects. This work was supported by Friedrich Miescher Institute funds, Seventh Framework Programme for Research (FP7) grants (RETICIRC, TREATRUSH, SEEBETTER, OPTONEURO) and a European Research Council grant to Bo.R. and a Marie Curie and EMBO fellowship to D.H., OM-00131/2007, OM-00132/2007, GOP-1.1.1-08/1-2008-0085, a grant of the Hungarian Academy of Sciences, Hungarian-French grant (TÉT_10-1-2011-0389) and Hungarian-Swiss grant (SH/7/2/8).

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Authors and Affiliations



Optical design was performed by P.M., G.S. and M.V. Software was written by G.K. In vitro measurements were performed by B.C., A.K., G.S. and Ba.R. In vivo measurements were designed by D.H. and performed by D.H., A.K., G.S. and Ba.R. Analysis was carried out by Ba.R., A.K., G.K. and G.S. This manuscript was written by Ba.R., Bo.R., D.H., G.K., A.K. and P.M., with comments from all authors. Ba.R., Bo.R., E.S.V. and P.M. supervised the project.

Corresponding author

Correspondence to Balázs Rózsa.

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Competing interests

G.K., E.S.V. and Ba.R. are owners of Femtonics and the patent WO2010076579.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Results 1–3, Supplementary Discussion, Supplementary Notes 1–9 and Supplementary Protocols 1–3 (PDF 17819 kb)

Supplementary Video 1

A 3D virtual reality environment for 3D two-photon imaging. This movie shows a surface-fitted pyramidal cell (located in the hippocampal CA1 region) and selected 3D measurement locations used to record the bAP-induced Ca2+ transients shown in Figure 2a. Using the 3D virtual reality environment, the 3D measurement locations can be freely modified or observed from any angle. Head-tracked shutter glasses ensure that the virtual objects maintain a stable, 'fixed' virtual position even when viewed from different viewpoints and angles. That is, the cell's virtual coordinate system is locked in space when the viewer's head position (view angle) changes; however, it can be rotated or shifted by the 3D 'bird' mouse. The bird also allows the 3D measurement points to be picked and repositioned in the virtual 3D space of the cell. (WMV 5934 kb)

Supplementary Video 2

Automatic selection of the measurement points for 3D two-photon imaging in vivo. This movie shows a bulk-loaded cell assembly located in the mouse visual cortex, visualized with real-time maximum-intensity projection in the 3D virtual-reality environment. After detecting putative neuron locations from the stack (see Supplementary Note 5), the experimenter can set the selection threshold with real-time control of the number of selected cells and their localization. (WMV 948 kb)

Supplementary Video 3

Millimeter-range image stack captured without mechanical movement. This movie shows a 3D image stack of neurons from a fluorescently labeled invertebrate ganglion also shown in Figure 1g. While capturing the images, the microscope objective was fixed; images were taken by AO z-focusing. The stack dimensions are 717 μm × 717 μm × 1,071 μm; 40 slices. (WMV 781 kb)

Supplementary Software 1

Use of AD9910. This summary contains information about the usage of the AD9910 DDS chip used to generate frequency signals for the acousto-optic crystals. Wiring to the FPGA, routines used to initialize the chip and Matlab code segments calculating the necessary register values during scanning are incorporated. (ZIP 254 kb)

Supplementary Software 2

A 3D interactive workstation module. This program provides a 3D VR environment with an open-source Matlab interface. It is possible to visualize and interact with 3D MIP projected volume data, surfaces and various annotation objects needed for controlling the experiments and for visualizing the results. It can perform mono or anaglyph views or be used in combination with the Leonar3Do virtual reality hardware. (ZIP 22535 kb)

Supplementary Software 3

Automatic drift-compensation algorithm. Description and code parts used for maintaining scan locations on the cells to measure. (ZIP 16 kb)

Supplementary Software 4

Automatic detection of fluorescently labeled cells. Matlab code identifying cell centers using three dimensional two-channel measurement data was developed for combined OGB-1 and SR-101 bolus loading experiments (Supplementary Note 9 and Supplementary Video 2). Accompanying sample data help evaluate the performance of the code. (ZIP 447 kb)

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Katona, G., Szalay, G., Maák, P. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat Methods 9, 201–208 (2012).

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