Mainen Z F, Maletic-Savatic M, Shi S H, Hayashi Y, Malinow R, Svoboda K
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Methods. 1999 Jun;18(2):231-9, 181. doi: 10.1006/meth.1999.0776.
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 key advantages can be easily implemented as a modification of commercial laser scanning microscopes.
双光子激发激光扫描显微镜（TPLSM）已成为完整神经组织中高分辨率荧光成像的首选工具。与其他光学技术相比，TPLSM能够以最小的光漂白和光毒性实现高分辨率成像并有效检测荧光信号。TPLSM的优势在诸如脑片等高度散射的环境中尤为明显。在此，我们描述了在活体脑片中对突触功能的各个方面进行成像的方法。为了将多种成像模式与膜片钳电生理记录相结合，我们发现定制一台直立显微镜很有好处。我们的设计目标主要是实验便利性和高效收集荧光。我们详细描述了我们的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融合蛋白的神经元的神经元形态动态测量，以及单个树突棘中钙动力学的功能成像。虽然我们的显微镜是定制仪器，但其关键优势可以很容易地作为商业激光扫描显微镜的一种改进来实现。
