Two-photon targeted patching (TPTP) in vivo
Two-photon targeted patching (TPTP) in vivo
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DOI：
10.1038/nprot.2006.100
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Margrie, Troy W.
中科院分区：
文献类型：
--
作者：
Komai, Shoji;Denk, Winfried;Margrie, Troy W.
双光子激发荧光激光扫描显微镜（2PLSM）提供了有关单细胞和群体水平生物过程时空特性的丰富信息。由于这种非线性光学方法允许在生物组织深处成像，因此 2PLSM 可以与膜片钳技术相结合，从体内特定荧光标记细胞获得电生理记录。这里，称为双光子靶向修补（TPTP）的方案描述了一种可用于记录完整动物中几乎被任何类型的荧光团标记的细胞的方法。我们的目标是使用在特定启动子控制下表达的绿色荧光蛋白（GFP）进行光学和遗传鉴定的神经元。因此，当 TPTP 与遗传方法相结合时，可以对特定神经元及其区室（包括树突）进行电生理记录。该技术可以在几个小时的过程中在相同的制剂中重复多次，并且同样适用于非神经元细胞类型。
Two-photon-excited fluorescence laser-scanning microscopy (2PLSM) has provided a wealth of information about the spatiotemporal properties of biological processes at the single cell and population level. Because such nonlinear optical methods allow for imaging deep within biological tissue, 2PLSM can be combined with patch-clamp techniques to obtain electrophysiological recordings from specific fluorescently labeled cells in vivo. Here a protocol referred to as two-photon targeted patching (TPTP) describes a method that may be used to record from cells in the intact animal labeled by virtually any type of fluorophore. We target neurons that have been optically and genetically identified using green fluorescent protein (GFP) expressed under the control of a specific promoter. TPTP when combined with genetic approaches therefore permits electrophysiological recordings from specified neurons and their compartments, including dendrites. This technique may be repeated in the same preparation many times over the course of several hours and is equally applicable to non-neuronal cell types.