Advanced Patch-Clamp Analysis for Neuroscientists pp 213-245

Part of the Neuromethods book series (NM, volume 113) | Cite as

Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo

Protocol

Abstract

Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 × 950 × 3000 μm3) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 × 290 × 200 μm3) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca2+ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca2+ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest.

Key words

Two-photon Acousto-optical Dendritic imaging Network imaging 3D Three-dimensional microscopy Angular dispersion compensation In vivo imaging Backpropagating action potentials Temporal super-resolution Dendritic spikes 3D scanning 3D virtual reality environment 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Balázs Rózsa
    • 1
    • 2
  • Gergely Szalay
    • 1
  • Gergely Katona
    • 1
    • 2
  1. 1.Institute of Experimental Medicine of the Hungarian Academy of SciencesBudapestHungary
  2. 2.Pázmány Péter UniversityBudapestHungary

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