原代培养小鼠皮质神经元中树突棘动态的活增强分辨率共焦成像方案。

Department of Physiology, Northwestern University, Chicago, IL 60611, USA.

Center for Advanced Microscopy, Northwestern University, Chicago, IL 60611, USA.

STAR Protoc. 2021 Apr 5;2(2):100427. doi: 10.1016/j.xpro.2021.100427. eCollection 2021 Jun 18.

Dendritic spinules are fine membranous protrusions of neuronal spines that play a role in synaptic plasticity, but their nanoscale requires resolution beyond conventional confocal microscopy, hindering live studies. Here, we describe how to track individual spinules in live dissociated cortical pyramidal neurons utilizing fluorescence labeling, optimized confocal imaging parameters, and post-acquisition iterative 3D deconvolution, employing NIS Elements software. This approach enables investigations of spinule structural dynamics and function without using super-resolution microscopy, which involves special fluorophores and/or high laser power. For complete details on the use and execution of this protocol, please refer to Zaccard et al. (2020).

树突棘刺是神经元棘突上的细微膜状突起，在突触可塑性中发挥作用，但它们的纳米尺度需要超越传统共聚焦显微镜的分辨率才能实现，这阻碍了活体研究。在这里，我们描述了如何利用荧光标记、优化的共聚焦成像参数以及基于 NIS Elements 软件的后期迭代 3D 反卷积，在活体分离的皮质锥体神经元中追踪单个棘刺。这种方法可以在不使用超分辨率显微镜的情况下研究棘刺的结构动力学和功能，超分辨率显微镜涉及特殊荧光染料和/或高激光功率。有关该方案的使用和执行的完整详细信息，请参考 Zaccard 等人（2020 年）。