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 KD 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 ([Fstim − Frest]/Frest), where Fstim is the intensity of the Ca2+ indicator in each image, and Frest is the intensity before nerve stimulation. Finally, various options for building a Ca2+-imaging rig are considered.