<em>In-vivo</em> and <em>Ex-vivo</em> Calcium Imaging of an Olfactory Circuit Neuron in the Third-Instar <em>Drosophila melanogaster</em> Larva

Calcium imaging is crucial for studying neural dynamics and brain function. However, current methods for imaging calcium in Drosophila larvae are challenging due to motion artifacts and specialized equipment needs. Here, we present a practical, low-cost method for performing both in vivo and ex vivo calcium imaging in third-instar Drosophila larvae, using a topical tissue adhesive for immobilizing samples. We also provide custom R scripts and a framework for data analysis, facilitating measurements of calcium changes under different experimental conditions. Using this approach, we successfully recorded calcium transients in a pair of larval CNS neurons with minimal motion artifacts, demonstrating reproducibility across preparations. The method is compatible with standard spinning disk confocal systems and can be adapted for DIY or commercial two-photon setups, making it accessible to laboratories with different resources. This approach may increase accessibility to larval calcium imaging studies and can be adapted to examine the effects of both external stimuli, such as odors, and internal stimuli, such as neuromodulators.