Abstract
Functional two-photon Ca2+-imaging is a versatile tool to study the dynamics of neuronal populations in brain slices and living animals. However, population imaging is typically restricted to a single two-dimensional image plane. By introducing an electrically tunable lens into the excitation path of a two-photon microscope we were able to realize fast axial focus shifts within 15 ms. The maximum axial scan range was 0.7 mm employing a 40x NA0.8 water immersion objective, plenty for typically required ranges of 0.2–0.3 mm. By combining the axial scanning method with 2D acousto-optic frame scanning and random-access scanning, we measured neuronal population activity of about 40 neurons across two imaging planes separated by 40 μm and achieved scan rates up to 20–30 Hz. The method presented is easily applicable and allows upgrading of existing two-photon microscopes for fast 3D scanning.
Highlights
Two-photon calcium imaging allows recording of neuronal activity in the intact brain down to depths of several hundred micrometers [1,2,3]
To estimate the axial range and optical resolution properties of the electrically tunable lens (ETL)-microscope objective combination, we modeled the complete acousto-optical deflectors (AODs) microscope including a 40x objective using ZEMAX
By combining the ETL as a fast axial scanning device with high-speed AOD-random access pattern scanning (RAPS) imaging, we demonstrated a versatile and expandable method for fast three-dimensional in vivo population imaging of neural networks
Summary
Two-photon calcium imaging allows recording of neuronal activity in the intact brain down to depths of several hundred micrometers [1,2,3]. For probing the activity of extended neuronal populations in the intact brain, 3D imaging is especially helpful, as the number of cells that can be recorded strongly increases with volume [4]. Single-trial recordings of neuronal network activity in 3D require fast imaging techniques to obtain a complete picture of local Ca2+-dynamics with high temporal resolution [4]. The ideal Ca2+-imaging method would allow three-dimensional measurements throughout hundreds of micrometers of tissue to sample large neuronal populations within a few milliseconds. Adding a third scan dimension exacerbates the challenge to record from neuronal populations with high sampling rates
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