RESUMO
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.
Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Movimento , Peixe-ZebraRESUMO
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.
Assuntos
Comportamento Animal/fisiologia , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Atividade Motora/fisiologia , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Potenciais de Ação/fisiologia , Animais , Córtex Cerebelar/citologia , Córtex Cerebelar/fisiologia , Dendritos/fisiologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Interneurônios/fisiologia , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Córtex Visual/citologia , Córtex Visual/fisiologiaRESUMO
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.
RESUMO
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.
Assuntos
Acústica/instrumentação , Desenho Assistido por Computador , Lentes , Luz , Modelos Teóricos , Espalhamento de Radiação , Simulação por Computador , Sistemas Microeletromecânicos/instrumentaçãoRESUMO
Remote focusing is widely used in 3D two-photon microscopy and 3D photostimulation because it enables fast axial scanning without moving the objective lens or specimen. However, due to the design constraints of microscope optics, remote focus units are often located in non-telecentric positions in the optical path, leading to significant depth-dependent 3D field distortions in the imaging volume. To address this limitation, we characterized 3D field distortions arising from non-telecentric remote focusing and present a method for distortion precompensation. We demonstrate its applicability for a 3D two-photon microscope that uses an acousto-optic lens (AOL) for remote focusing and scanning. We show that the distortion precompensation method improves the pointing precision of the AOL microscope to < 0.5 µm throughout the 400 × 400 × 400 µm imaging volume.
RESUMO
We describe a high speed 3D Acousto-Optic Lens Microscope (AOLM) for femtosecond 2-photon imaging. By optimizing the design of the 4 AO Deflectors (AODs) and by deriving new control algorithms, we have developed a compact spherical AOL with a low temporal dispersion that enables 2-photon imaging at 10-fold lower power than previously reported. We show that the AOLM can perform high speed 2D raster-scan imaging (>150 Hz) without scan rate dependent astigmatism. It can deflect and focus a laser beam in a 3D random access sequence at 30 kHz and has an extended focusing range (>137 mum; 40X 0.8NA objective). These features are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space.
Assuntos
Imageamento Tridimensional , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Algoritmos , Desenho de Equipamento , Modelos Teóricos , Pólen/ultraestrutura , Transdutores , UltrassomRESUMO
BACKGROUND: Two-photon microscopy is widely used to study brain function, but conventional microscopes are too slow to capture the timing of neuronal signalling and imaging is restricted to one plane. Recent development of acousto-optic-deflector-based random access functional imaging has improved the temporal resolution, but the utility of these technologies for mapping 3D synaptic activity patterns and their performance at the excitation wavelengths required to image genetically encoded indicators have not been investigated. NEW METHOD: Here, we have used a compact acousto-optic lens (AOL) two-photon microscope to make high speed [Ca(2+)] measurements from spines and dendrites distributed in 3D with different excitation wavelengths (800-920 nm). RESULTS: We show simultaneous monitoring of activity from many synaptic inputs distributed over the 3D arborisation of a neuronal dendrite using both synthetic as well as genetically encoded indicators. We confirm the utility of AOL-based imaging for fast in vivo recordings by measuring, simultaneously, visually evoked responses in 100 neurons distributed over a 150 µm focal depth range. Moreover, we explore ways to improve the measurement of timing of neuronal activation by choosing specific regions within the cell soma. COMPARISON WITH EXISTING METHODS: These results establish that AOL-based 3D random access two-photon microscopy has a wider range of neuroscience applications than previously shown. CONCLUSIONS: Our findings show that the compact AOL microscope design has the speed, spatial resolution, sensitivity and wavelength flexibility to measure 3D patterns of synaptic and neuronal activity on individual trials.