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