title: Imaging and analysis of nonratiometric calcium indicators at the Drosophila larval neuromuscular junction.
abstract: 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.
authors: Gregory T
authors: GT
authors: Macleod
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis*; D004330:Drosophila / Q000737:chemistry*; D004772:Entomology / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007202:Indicators and Reagents; D007814:Larva / Q000737:chemistry; D009446:Neurobiology / Q000379:methods*; D009469:Neuromuscular Junction / Q000737:chemistry*; D013048:Specimen Handling / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D002118:Calcium