■双光子光遗传学将非线性激发与神经元的非侵入性激活相结合,从而能够以高度的空间精度操纵神经回路。结合双光子群体钙成像,这些方法包括一个灵活的平台,用于神经回路的全光学询问。然而,许多光学和生物学因素决定了这种方法在体内的精确精度,最有用的应用。
■我们旨在评估光学点扩散函数(OPSF)如何为神经生物学中的双光子光刺激的空间精度做出贡献。
■我们使用空间光调制器改变了光刺激光束的OPSF的轴向扩展。随后,钙成像用于监测小鼠新皮层2层神经元双光子光刺激的轴向空间精度。
■我们发现光学分辨率并不总是双光子光遗传光刺激的空间精度的限制因素,通过这样做,揭示了实现最大精度必须改进的关键因素。
■我们的结果通过以以前未报道的方式提供受控实验的关键见解,使未来的工作能够专注于最佳因素。这项研究可以应用于推进全光学询问的最新技术,扩展用于神经科学研究的工具包,以在神经回路运行的关键水平上实现时空精度。
UNASSIGNED: Two-photon optogenetics combines nonlinear excitation with noninvasive activation of neurons to enable the manipulation of neural circuits with a high degree of spatial precision. Combined with two-photon population calcium imaging, these approaches comprise a flexible platform for all-optical interrogation of neural circuits. However, a multitude of optical and biological factors dictate the exact precision of this approach in vivo, where it is most usefully applied.
UNASSIGNED: We aimed to assess how the optical point spread function (OPSF) contributes to the spatial precision of two-photon photostimulation in neurobiology.
UNASSIGNED: We altered the axial spread of the OPSF of the photostimulation beam using a spatial light modulator. Subsequently, calcium imaging was used to monitor the axial spatial precision of two-photon photostimulation of layer 2 neurons in the mouse neocortex.
UNASSIGNED: We found that optical resolution is not always the limiting factor of the spatial precision of two-photon optogenetic photostimulation and, by doing so, reveal the key factors that must be improved to achieve maximal precision.
UNASSIGNED: Our results enable future work to focus on the optimal factors by providing key insight from controlled experiments in a manner not previously reported. This research can be applied to advance the state-of-the-art of all-optical interrogation, extending the toolkit for neuroscience research to achieve spatiotemporal precision at the crucial levels in which neural circuits operate.