Department of Physics, Princeton University, Princeton, New Jersey, USA.
Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
Nat Methods. 2017 Apr;14(4):420-426. doi: 10.1038/nmeth.4226. Epub 2017 Mar 20.
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.
使用荧光指示剂的双光子激光扫描显微镜对钙动力学进行研究是一种广泛用于体内大规模记录神经活动的成像方法。在这里，我们介绍了使用立体视觉的神经元容积双光子成像（vTwINS），这是一种容积钙成像方法，它使用拉长的 V 形点扩散函数来对 3D 脑体积进行成像。单个神经元在产生的 2D 图像中投射到空间上偏移的“图像对”，并且投射之间的分离距离与体积中的深度成正比。为了对单个神经元的荧光时间序列进行解混，我们引入了一种改进的正交匹配追踪算法，该算法还可以推断 3D 体积内的源位置。我们通过对小鼠初级视觉皮层和海马体的神经群体活动进行成像来展示 vTwINS。我们的结果表明，vTwINS 提供了一种有效的容积双光子钙成像方法，在保持高帧率的同时增加了记录的神经元数量。
