ABSTRACT
Two-photon microscopy is widely used to investigate brain function across multiple spatial scales. However, measurements of neural activity are compromised by brain movement in behaving animals. Brain motion-induced artifacts are typically corrected using post hoc processing of two-dimensional images, but this approach is slow and does not correct for axial movements. Moreover, the deleterious effects of brain movement on high-speed imaging of small regions of interest and photostimulation cannot be corrected post hoc. To address this problem, we combined random-access three-dimensional (3D) laser scanning using an acousto-optic lens and rapid closed-loop field programmable gate array processing to track 3D brain movement and correct motion artifacts in real time at up to 1 kHz. Our recordings from synapses, dendrites and large neuronal populations in behaving mice and zebrafish demonstrate real-time movement-corrected 3D two-photon imaging with submicrometer precision.
Subject(s)
Imaging, Three-Dimensional/methods , Microscopy, Fluorescence, Multiphoton/methods , Animals , Female , Male , Mice , Mice, Inbred C57BL , Movement , ZebrafishABSTRACT
Understanding how neural circuits process information requires rapid measurements of activity from identified 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.
Subject(s)
Behavior, Animal/physiology , Imaging, Three-Dimensional/methods , Microscopy, Fluorescence, Multiphoton/methods , Motor Activity/physiology , Neurons/physiology , Voltage-Sensitive Dye Imaging/methods , Action Potentials/physiology , Animals , Cerebellar Cortex/cytology , Cerebellar Cortex/physiology , Dendrites/physiology , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Interneurons/physiology , Mice , Mice, Transgenic , Patch-Clamp Techniques , Pyramidal Cells/physiology , Visual Cortex/cytology , Visual Cortex/physiologyABSTRACT
Acousto-optic deflectors (AODs) arranged in series and driven with linearly chirped frequencies can rapidly focus and tilt optical wavefronts, enabling high-speed 3D random access microscopy. Non-linearly chirped acoustic drive frequencies can also be used to shape the optical wavefront allowing a range of higher-order aberrations to be generated. However, to date, wavefront shaping with AODs has been achieved by using single laser pulses for strobed illumination to 'freeze' the moving acoustic wavefront, limiting voxel acquisition rates. Here we show that dynamic wavefront shaping can be achieved by applying non-linear drive frequencies to a pair of AODs with counter-propagating acoustic waves, which comprise a cylindrical acousto-optic lens (AOL). Using a cylindrical AOL we demonstrate high-speed continuous axial line scanning and the first experimental AOL-based correction of a cylindrical lens aberration at 30 kHz, accurate to 1/35th of a wave at 800 nm. Furthermore, we develop a model to show how spherical aberration, which is the major aberration in AOL-based remote-focusing systems, can be partially or fully corrected with AOLs consisting of four or six AODs, respectively.
ABSTRACT
A spherical acousto-optic lens (AOL) consists of four acousto-optic deflectors (AODs) that can rapidly and precisely control the focal position of an optical beam in 3D space. Development and application of AOLs has increased the speed at which 3D random access point measurements can be performed with a two-photon microscope. This has been particularly useful for measuring brain activity with fluorescent reporter dyes because neuronal signalling is rapid and sparsely distributed in 3D space. However, a theoretical description of light propagation through AOLs has lagged behind their development, resulting in only a handful of simplified principles to guide AOL design and optimization. To address this we have developed a ray-based computer model of an AOL incorporating acousto-optic diffraction and refraction by anisotropic media. We extended an existing model of a single AOD with constant drive frequency to model a spherical AOL: four AODs in series driven with linear chirps. AOL model predictions of the relationship between optical transmission efficiency and acoustic drive frequency including second order diffraction effects closely matched experimental measurements from a 3D two-photon AOL microscope. Moreover, exploration of different AOL drive configurations identified a new simple rule for maximizing the field of view of our compact AOL design. By providing a theoretical basis for understanding optical transmission through spherical AOLs, our open source model is likely to be useful for comparing and improving different AOL designs, as well as identifying the acoustic drive configurations that provide the best transmission performance over the 3D focal region.
