Content area
Full Text
BRIEF COMMUNICATIONS
Random-access scanning microscopy for 3D imaging in awake behaving animals
K M Naga Srinivas Nadella, Hana Ro, Chiara Baragli, Victoria A Griffiths, George Konstantinou,
Theo Koimtzis, Geoffrey J Evans, Paul A Kirkby &
R Angus Silver
Understanding how neural circuits process information requires rapid measurements of activity from identied neurons distributed in 3D space. Here we describe an acousto-optic lens two-photon microscope that performs high-speed focusing and line scanning within a volume spanning hundreds of micrometers. We demonstrate its random-access functionality by selectively imaging cerebellar interneurons sparsely distributed in 3D space and by simultaneously recording from the soma, proximal and distal dendrites of neocortical pyramidal cells in awake behaving mice.
Understanding information processing in the brain requires the measurement of signals as they flow rapidly through neuronal circuits deep within scattering tissue. However, circuits are often arranged in layers, neuronal subtypes are sparsely distributed, and dendrites and axons radiate and branch in 3D space. Since neuronal compartments of interest often occupy a small fraction of total tissue volume, considerably higher temporal resolution could be achieved if, rather than imaging the whole volume, regions of interest (ROIs) were selectively imaged. Although conventional resonant galvanometer two-photon laser scanners can image rapidly1 and achieve fast focusing with electrically tuneable2 or ultrasound lenses3 or with a pair of matched objective lenses and a small mirror4, their inertia limits the speed at which they can jump between ROIs. This makes it difficult to monitor sparsely distributed neuronal activity in 3D with conventional imaging methods.
Acousto-optic lens (AOL) microscopes57 can perform inertia-free focusing, thereby enabling acquisition of 3D random-access multiphoton (3D-RAMP) point measurements within an imaging volume at 3550 kHz (refs. 710). However, the major technical limitation of acousto-optic-deflector (AOD)-based scanning devices, including conventional AOLs, is that they cannot perform full-frame continuous line scanning away from the natural focal plane of the objective6,11,12 (Supplementary Notes 1 and 2). This is problematic for in vivo imaging because the acquisition
Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK. Correspondence should be addressed to R.A.S. ([email protected]).
RECEIVED 24 MAY; ACCEPTED 14 SEPTEMBER; PUBLISHED ONLINE 17 OCTOBER 2016; http://dx.doi.org/10.1038/nmeth.4033
Web End =DOI:10.1038/NMETH.4033
of z-stacks is slow using 3D-RAMP voxel-by-voxel pointing7 or miniscans (consisting of a few voxels)9 to form each...