Volumetric Two-photon Imaging of Neurons Using Stereoscopy (vTwINS)

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 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.

A Guide to Emerging Technologies for Large-Scale and Whole-Brain Optical Imaging of Neuronal Activity

“…An interesting approach where a different type of engineered PSF was used is vTwINS (Song et al 2017). Here, a volume was imaged using an elongated, V-shaped PSF (Fig.…”

Bessel beam tomography for fast volume imaging

“…Depending on the parameters and methods used to generate Bessel beams, different profiles in terms of width, length and axial intensity distribution result [21]. The axial intensity profile additionally depends on the placement of the collimating lenses [12,21]. Here, we aimed for a beam length of about 80 µm as a compromise between recording a sufficiently large volume while limiting the power required for two-photon excitation in each beam.…”

“…Combined with camera detectors, these approaches work best for weakly scattering samples [3,4], but Bessel beams or line foci [5] can also be used for imaging with non-descanned PMT detection. This is more suitable for imaging in scattering tissue and results in a projection of the entire imaging volume along the beam axis [8][9][10][11][12]. While for Bessel beams this approach offers the advantage of recording from an entire volume in a single frame scan, it comes at the cost of loosing all axial resolution.…”

“…Axial resolution was restored in experiments that used two Bessel beams which imaged the sample at different angles [9,10,12] for generating two different projections. These projections were either recorded sequentially [10] or simultaneously (as required for moving samples such as brain tissue in vivo) in a single channel by averaging over two different projections [12]. From these projections axial information was recovered based on the distance between prominent, sparse features such as fluorescent beads or cell bodies [10,12].…”

Volumetric two-photon imaging in live cells and embryos via axially gradient excitation

“…However, this methodology can only provide 2D projection images of the samples and lost the information of axial location of fluorophores. Another strategy, volumetric two-photon imaging of neurons using stereoscopy (vTwINS) 12 , used an elongated, V-shaped PSF to encode the depth location of neurons into the separation distance between their 2D projective image pairs. This method can recorded the neuron activity within a 45 μm thick volume at a 30 Hz while preserving the depth location of somas.…”