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Nat Methods. 2017 Apr;14(4):420-426. doi: 10.1038/nmeth.4226. Epub 2017 Mar 20.

Volumetric two-photon imaging of neurons using stereoscopy (vTwINS).

Author information

1
Department of Physics, Princeton University, Princeton, New Jersey, USA.
2
Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
3
Bezos Center for Neural Circuit Dynamics, Princeton University, Princeton, New Jersey, USA.
4
Department of Psychology, Princeton University, Princeton, New Jersey, USA.
5
Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.

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.

PMID:
28319111
PMCID:
PMC5551981
DOI:
10.1038/nmeth.4226
[Indexed for MEDLINE]
Free PMC Article

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