Volumetric two-photon imaging of neurons using stereoscopy (vTwINS). Author Alexander Song, Adam Charles, Sue Koay, Jeff Gauthier, Stephan Thiberge, Jonathan Pillow, David Tank Publication Year 2017 Type Journal Article Abstract Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large-scale recording of neural activity in vivo. Here, we introduce volumetric two-photon imaging of neurons using stereoscopy (vTwINS), a volumetric calcium imaging method that uses an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced 'image pairs' in the resulting 2D image, and the separation distance between projections is proportional to depth in the volume. To demix the fluorescence time series of individual neurons, we introduce a modified orthogonal matching pursuit algorithm that also infers source locations within the 3D volume. We illustrated vTwINS by imaging neural population activity in the mouse primary visual cortex and hippocampus. Our results demonstrated that vTwINS provides an effective method for volumetric two-photon calcium imaging that increases the number of neurons recorded while maintaining a high frame rate. Keywords Animals, Calcium, Female, Male, Neurons, Molecular Imaging, Visual Cortex, Algorithms, Mice, Transgenic, Hippocampus, Microscopy, Confocal, Imaging, Three-Dimensional, Microscopy, Fluorescence, Multiphoton Journal Nat Methods Volume 14 Issue 4 Pages 420-426 Date Published 2017 Apr ISSN Number 1548-7105 DOI 10.1038/nmeth.4226 Alternate Journal Nat Methods PMCID PMC5551981 PMID 28319111 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML