Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo
Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo
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DOI：
10.1038/nprot.2006.58
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Konnerth, Arthur
中科院分区：
文献类型：
--
作者：
Garaschuk, Olga;Milos, Ruxandra-Iulia;Konnerth, Arthur
现代神经科学面临的挑战之一是了解体内神经元协同功能的规则。这个问题可以使用诸如双光子显微镜等非侵入性高分辨率成像技术来解决。本实验方案描述了一种对用膜透性荧光指示剂染料染色的神经网络进行体内双光子钙成像的通用方法。它基于将染料靶向压力喷射到感兴趣的组织中，并且可用于多种指示剂染料，包括俄勒冈绿488 BAPTA - 1乙酰氧基甲酯和Fura - 2乙酰氧基甲酯。通过使用染料混合物和多色成像，该技术能够可视化大脑表面以下多达500微米的不同神经元和神经胶质细胞。它适用于对所有发育阶段的各种不同物种（例如小鼠、大鼠、猫和斑马鱼）的脑组织进行染色。当与脑显微内窥镜结合时，它能够监测清醒且有行为的动物的细胞内钙信号。执行该实验方案所需的总时间，包括解剖和细胞染色，约为2小时。此后，成像实验可能至少进行6小时。
One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 mu m below the brain surface. It is suitable for staining the brain tissue of various different species (e. g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is similar to 2 h. Thereafter, imaging experiments might be performed for at least 6 h.