Calcium imaging using two-photon scanning microscopy has become an essential tool in neuroscience. However, in its typical implementation, the tradeoffs between fields of view, acquisition speeds, and depth restrictions in scattering brain tissue pose severe limitations. Here, using an integrated systems-wide optimization approach combined with multiple technical innovations, we introduce a new design paradigm for optical microscopy based on maximizing biological information while maintaining the fidelity of obtained neuron signals. Our modular design utilizes hybrid multi-photon acquisition and allows volumetric recording of neuroactivity at single-cell resolution within up to 1 3 1 3 1.22 mm volumes at up to 17 Hz in awake behaving mice. We establish the capabilities and potential of the different configurations of our imaging system at depth and across brain regions by applying it to in vivo recording of up to 12,000 neurons in mouse auditory cortex, posterior parietal cortex, and hippocampus.
We consider the general problem of free-space beam shaping for coupling in and out of higher order modes (HOMs) in optical fibers with high purity and low loss. We compare the performance of two simple phase structures - binary phase plates (BPPs) and axicons - for converting Gaussian beams to HOMs and vice versa. Both axicons and BPPs allow for excitation of modes with high purity (>15 dB parasitic mode suppression), or conversion of HOMs to near-Gaussian beams (M2 < 1.25). Axicon coupling in single-clad fibers allows for lower loss (0.85 ± 0.1 dB) conversion than BPPs (1.7 ± 0.1 dB); but BPPs are compatible with any fiber design, and allow for rapid switching between modes. The experiments detailed here use all commercial components and fibers, allowing for a simple means to investigate the unique properties of multi-mode fibers.
Two-photon microscopy together with genetically encodable calcium indicators has emerged as a standard tool for high-resolution imaging of neuroactivity in scattering brain tissue. However, its various realizations have not overcome the inherent tradeoffs between speed and spatiotemporal sampling in a principled manner which would be necessary to enable, amongst other applications, mesoscale volumetric recording of neuroactivity at cellular resolution and speed compatible with resolving calcium transients. Here, we introduce Light Beads Microscopy (LBM), a scalable and spatiotemporally optimal acquisition approach limited only by fluorescence life-time, where a set of axially-separated and temporally-distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 x 10^8 voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple scales in the mouse cortex, including cellular resolution recordings within ~3 x 5 x 0.5 mm^3 volumes containing >200,000 neurons at ~5 Hz, recording of populations of ~1 million neurons within ~5.4 x 6 x 0.5 mm^3 volumes at ~2 Hz as well as higher-speed (9.6 Hz) sub-cellular resolution volumetric recordings. LBM provides an unprecedented opportunity for discovering the neurocomputations underlying cortex-wide encoding and processing of information in the mammalian brain.
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