RT Journal Article SR Electronic T1 Volumetric Two-photon Imaging of Neurons Using Stereoscopy (vTwINS) JF bioRxiv FD Cold Spring Harbor Laboratory SP 073742 DO 10.1101/073742 A1 Alexander Song A1 Adam S. Charles A1 Sue Ann Koay A1 Jeff L. Gauthier A1 Stephan Y. Thiberge A1 Jonathan W. Pillow A1 David W. Tank YR 2016 UL http://biorxiv.org/content/early/2016/09/06/073742.abstract AB 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 employs 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 images is proportional to depth in the volume. To demix the fluorescence time series of individual neurons, we introduce a novel orthogonal matching pursuit algorithm that also infers source locations within the 3D volume. We illustrate vTwINS by imaging neural population activity in mouse primary visual cortex and hippocampus. Our results demonstrate 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.