Imaging and Analysis of Nonratiometric Calcium Indicators at the Drosophila Larval Neuromuscular Junction

Abstract

Ca²⁺ indicators can be loaded into a Drosophila larval neuromuscular junction (NMJ) preparation using several methods, including topical application of membrane-permeant Ca²⁺ indicators, forward-filling of dextran conjugates, and direct injection. This article describes how such an NMJ preparation loaded with Ca²⁺ 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 Ca²⁺ indicator must be quantified to obtain an estimate of the change in the concentration of free Ca²⁺ (Δ[Ca²⁺]). 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 Ca²⁺ indicator is in excess of the maximum value of [Ca²⁺] during the response, then ΔF/F is considered to be linearly related to Δ[Ca²⁺]. 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 Ca²⁺ indicator in each image, and F_rest is the intensity before nerve stimulation. Finally, various options for building a Ca²⁺-imaging rig are considered.