Two-photon imaging in living brain slices
Two-photon imaging in living brain slices
复制标题
DOI：
10.1006/meth.1999.0776
复制
发表时间：
1999-06-01
期刊：
影响因子：
4.8
通讯作者：
Svoboda, K
中科院分区：
文献类型：
--
作者：
Mainen, ZF;Maletic-Savatic, M;Svoboda, K
双光子激发激光扫描显微镜（TPLSM）已成为完整神经组织高分辨率荧光成像的首选工具。与其他光学技术相比，TPLSM可以实现高分辨率成像和高效检测荧光信号，并且光漂白和光毒性最小。TPLSM的优点在高散射环境中尤其明显，例如脑切片。在这里，我们描述了我们的方法成像突触功能的各个方面在活的大脑切片。为了将几种成像模式与膜片钳电生理记录结合起来，我们发现定制一个直立显微镜是有利的。我们的设计目标主要是实验方便和有效收集荧光。详细介绍了TPLSM成像系统及其性能。我们展示了表达绿色荧光蛋白（GFP）和GFP融合蛋白的神经元形态学的动态测量，以及单个树突棘钙动力学的功能成像。虽然我们的显微镜是一种定制仪器，但它的优点可以很容易地作为商用激光扫描显微镜的改进而实现。(C) 1999学术出版社。
Two-photon excitation laser scanning microscopy (TPLSM) has become the tool of choice for high-resolution fluorescence imaging in intact neural tissues. Compared with other optical techniques, TPLSM allows high-resolution imaging and efficient detection of fluorescence signal with minimal photobleaching and phototoxicity. The advantages of TPLSM are especially pronounced in highly scattering environments such as the brain slice. Here we describe our approaches to imaging various aspects of synaptic function in living brain slices. To combine several imaging modes together with patch-clamp electrophysiological recordings we found it advantageous to custom-build an upright microscope. Our design goals were primarily experimental convenience and efficient collection of fluorescence. We describe our TPLSM imaging system and its performance in detail. We present dynamic measurements of neuronal morphology of neurons expressing green fluorescent protein (GFP) and GFP fusion proteins as well as functional imaging of calcium dynamics in individual dendritic spines. Although our microscope is a custom instrument, its hey advantages can be easily implemented as a modification of commercial laser scanning microscopes. (C) 1999 Academic Press.