Macleod Gregory T
Cold Spring Harb Protoc. 2012 Jul 1;2012(7):802-9. doi: 10.1101/pdb.prot070110.
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+) (Δ[Ca(2+)]). The change in intensity is conventionally represented as the expression "ΔF/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 ΔF/F is considered to be linearly related to Δ[Ca(2+)]. In practice, ΔF/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.
可以使用多种方法将钙离子（Ca²⁺）指示剂加载到果蝇幼虫神经肌肉接头（NMJ）标本中，包括局部应用膜通透性Ca²⁺指示剂、葡聚糖缀合物的正向填充和直接注射。本文描述了如何设置加载有Ca²⁺指示剂的NMJ标本，以便在刺激其支配神经细胞时对肌纤维进行成像。提供了一个简单的方案，用于收集和分析一组成像数据，以及图像分析中涉及的计算顺序。必须对Ca²⁺指示剂强度的变化进行量化，以获得游离Ca²⁺浓度变化（Δ[Ca²⁺]）的估计值。强度变化通常用“ΔF/F”表示。简单地说，这是相对于静息荧光强度的荧光强度变化。如果Ca²⁺指示剂的解离常数（Kₐ）在反应过程中超过[Ca²⁺]的最大值，则认为ΔF/F与Δ[Ca²⁺]呈线性关系。在实践中，使用简单算法（[F（刺激）-F（静息）]/F（静息））为每个图像计算ΔF/F，其中F（刺激）是每个图像中Ca²⁺指示剂的强度，F（静息）是神经刺激前的强度。最后，考虑了构建Ca²⁺成像装置的各种选项。
