Department of Neuroscience, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Department of Imaging Physics, Delft University of Technology, Delft, the Netherlands.
Nat Protoc. 2019 Mar;14(3):864-900. doi: 10.1038/s41596-018-0118-2. Epub 2019 Feb 25.
Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammalian origin by use of photoswitches, visible light (typically), and genetic modification. Thus, synthetic optogenetics facilitates interrogation of native neuronal signaling mechanisms. However, the poor tissue penetration of visible wavelengths impedes the use of the technique in tissue, as two-photon excitation (2PE) is typically required to access the near-infrared window. Here, we describe an alternative technique that uses 2PE-compatible photoswitches (section 1) for photoactivation of genetically modified glutamate receptors (section 2). Furthermore, for fast, multi-region photoactivation, we describe the use of 2P-digital holography (2P-DH) (section 3). We detail how to combine 2P-DH and synthetic optogenetics with electrophysiology, or with red fluorescence Ca recordings, for all-optical neural interrogation. The time required to complete the methods, aside from obtaining the necessary reagents and illumination equipment, is ~3 weeks.
光遗传学工具为用户提供了光控细胞活动的能力。通常，通过表达来自光合生物的蛋白质来实现激活，例如微生物视蛋白（例如 ChR2）。或者，一种类似的方法，即合成光遗传学，通过使用光开关、可见光（通常）和基因修饰来实现对哺乳动物来源的蛋白质的光控。因此，合成光遗传学促进了对天然神经元信号机制的研究。然而，可见光波长的组织穿透率差阻碍了该技术在组织中的应用，因为通常需要双光子激发（2PE）来进入近红外窗口。在这里，我们描述了一种替代技术，该技术使用 2PE 兼容的光开关（第 1 节）来对基因修饰的谷氨酸受体进行光激活（第 2 节）。此外，对于快速、多区域光激活，我们描述了使用双光子数字全息术（2P-DH）（第 3 节）的情况。我们详细介绍了如何将 2P-DH 和合成光遗传学与电生理学或红色荧光 Ca 记录相结合，进行全光学神经检测。除了获得必要的试剂和照明设备外，完成这些方法所需的时间约为 3 周。
