Imaging and analysis of nonratiometric calcium indicators at the Drosophila larval neuromuscular junction.
Imaging and analysis of nonratiometric calcium indicators at the Drosophila larval neuromuscular junction.
复制标题
DOI：
10.1101/pdb.prot070110
复制
发表时间：
2012-07-01
影响因子：
--
通讯作者：
Macleod, Gregory T
中科院分区：
文献类型：
--
作者：
Macleod, Gregory T
Ca(2+) 指示剂可以使用多种方法加载到果蝇幼虫神经肌肉接头 (NMJ) 制剂中，包括局部应用膜渗透性 Ca(2+) 指示剂、葡聚糖缀合物的前向填充和直接注射。本文介绍了如何设置装载 Ca(2+) 指示剂的 NMJ 制剂，以在刺激其支配神经细胞期间对肌纤维进行成像。提供了一个简单的协议来收集和分析一组成像数据，以及图像分析中涉及的计算序列。必须对 Ca(2+) 指示剂强度的变化进行量化，以获得游离 Ca(2+) 浓度变化的估计值 (Delta[Ca(2+)])。强度的变化通常表示为“DeltaF/F”。简单地说，这是荧光强度相对于静息荧光强度的变化。如果Ca(2+)指标的K(D)在响应期间超过[Ca(2+)]的最大值，则DeltaF/F被认为与Delta[Ca(2+)]线性相关。实际上，使用简单的算法（[F(stim) - F(rest)]/F(rest)）计算每幅图像的 DeltaF/F，其中 F(stim) 是每幅图像中 Ca(2+) 指示剂的强度，F(rest) 是神经刺激前的强度。最后，考虑了构建 Ca(2+) 成像装置的各种选项。
Ca(2+) indicators can be loaded into a Drosophila larval neuromuscular junction (NMJ) preparation using several methods, including topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. This article describes how such an NMJ preparation loaded with Ca(2+) indicator is set up for imaging of the muscle fiber during stimulation of its innervating nerve cell. A simple protocol is provided for collecting and analyzing a set of imaging data, together with the sequence of calculations involved in image analysis. The change in the intensity of the Ca(2+) indicator must be quantified to obtain an estimate of the change in the concentration of free Ca(2+) (Delta[Ca(2+)]). The change in intensity is conventionally represented as the expression "DeltaF/F." Simply put, this is the change in fluorescence intensity relative to the resting fluorescence intensity. If the K(D) of the Ca(2+) indicator is in excess of the maximum value of [Ca(2+)] during the response, then DeltaF/F is considered to be linearly related to Delta[Ca(2+)]. In practice, DeltaF/F is calculated for each image using a simple algorithm ([F(stim) - F(rest)]/F(rest)), where F(stim) is the intensity of the Ca(2+) indicator in each image, and F(rest) is the intensity before nerve stimulation. Finally, various options for building a Ca(2+)-imaging rig are considered.