Neural anatomy and optical microscopy (NAOMi) simulation for evaluating calcium imaging methods.

BACKGROUND: The past decade has seen a multitude of new in vivo functional imaging methodologies. However, the lack of ground-truth comparisons or evaluation metrics makes the large-scale, systematic validation vital to the continued development and use of optical microscopy impossible. NEW-METHOD: We provide a new framework for evaluating two-photon microscopy methods via in silico Neural Anatomy and Optical Microscopy (NAOMi) simulation. Our computationally efficient model generates large anatomical volumes of mouse cortex, simulates neural activity, and incorporates optical propagation and scanning to create realistic calcium imaging datasets. RESULTS: We verify NAOMi simulations against in vivo two-photon recordings from mouse cortex. We leverage this in silico ground truth to directly compare different segmentation algorithms and optical designs. We find modern segmentation algorithms extract strong neural time-courses comparable to estimation using oracle spatial information, but with an increase in the false positive rate. Comparison between optical setups demonstrate improved resilience to motion artifacts in sparsely labeled samples using Bessel beams, increased signal-to-noise ratio and cell-count using low numerical aperture Gaussian beams and nuclear GCaMP, and more uniform spatial sampling with temporal focusing versus multi-plane imaging. COMPARISON WITH EXISTING METHODS: NAOMi is a first-of-its kind framework for assessing optical imaging modalities. Existing methods are either anatomical simulations or do not address functional imaging. Thus there is no competing method for simulating realistic functional optical microscopy data. CONCLUSIONS: By leveraging the rich accumulated knowledge of neural anatomy and optical physics, we provide a powerful new tool to assess and develop important methods in neural imaging.

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

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.