Matsuzaki Masanori, Kasai Haruo
Cold Spring Harb Protoc. 2011 May 1;2011(5):pdb.prot5620. doi: 10.1101/pdb.prot5620.
Two-photon uncaging takes advantage of the inherent optical sectioning power of two-photon excitation to generate highly localized concentration increases of neurotransmitters such as glutamate. This can be used to activate isolated clusters of receptors and, thus, produce maps of receptor densities, or activate intracellular signal transduction under these receptors, in three dimensions and in complex structures such as hippocampal brain slices. Used in combination with two-photon imaging, two-photon uncaging provides a means to study the long-term structural and functional consequences of stimulation of structures such as dendritic spines. This protocol gives an overview of the procedures used for two-photon uncaging microscopy. It includes a detailed description of the development of a microscope that enables effective two-photon release of caged neurotransmitters and provides several examples of its use in cultured and acutely isolated hippocampal neurons.
双光子解笼利用双光子激发固有的光学切片能力，使诸如谷氨酸等神经递质的浓度在高度局部化区域内升高。这可用于激活孤立的受体簇，从而生成受体密度图，或在三维空间以及诸如海马脑片这样的复杂结构中激活这些受体下的细胞内信号转导。与双光子成像结合使用时，双光子解笼提供了一种手段，用于研究诸如树突棘等结构受到刺激后的长期结构和功能后果。本方案概述了双光子解笼显微镜所使用的程序。它详细描述了一台显微镜的开发过程，该显微镜能够有效地实现笼状神经递质的双光子释放，并提供了其在培养的和急性分离的海马神经元中应用的几个示例。
