KEYWORDS: In vivo imaging, Light sources and illumination, Confocal microscopy, Signal to noise ratio, Optical imaging, Neuroimaging, Crosstalk, Brain, Tissues, Tissue optics
Advancements in genetically encoded voltage indicators (GEVIs) have made it possible to measure cellular membrane potential changes optically. But performing GEVI imaging in vivo remains highly challenging due to factors such as low GEVI concentrations, modest signal dynamic range, tissue scattering, out-of-focus fluorescence. To address these challenges, we developed a microscopy technique that take advatanges from both widefield targeted illumination and confocal background rejection, enabling high SNR low crosstalk GEVI imaging across millimeter fields-of-view, at supra-kilohertz frame rates, over extended durations, and at high penetration depths. We demonstrate our technique under a variety of imaging conditions across multiple brain regions and with different classes of GEVIs.
KEYWORDS: Optical imaging, Confocal microscopy, Stereoscopy, 3D image processing, In vivo imaging, Signal to noise ratio, Scattering, Microscopy, Microscopes, In vitro testing
Fluorescent genetically encoded voltage indicators can be combined with optical imaging to provide high-throughput electrophysiologic recordings with single-spike resolution and subthreshold sensitivity. Such voltage imaging is highly demanding in terms of signal collection; thus, most experiments have been performed with widefield one-photon microscopy. Unfortunately, widefield techniques are susceptible to out-of-focus background and scattering, which degrades SNR, especially in high-density slice or in vivo experiments. In this work, we describe a multi-plane near-kHz-rate confocal microscope that effectively suppresses undesired background. This technique enables more densely labeled in vitro and in vivo imaging experiments, critical for the dissection of neural circuit dynamics.
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