Live imaging and quantitation of insect feeding-induced Ca2+ signal using GCaMP3-based system in Nicotiana benthamiana

Summary Wounding evokes transient increases in cytosolic calcium (Ca 2+ ) concentration. Visualizing real-time Ca 2+ flux provides new insights into Ca 2+ -signaling pathways. Here, we outline a protocol to detect insect feeding-induced Ca 2+ flux elevation in Nicotiana benthamiana leaves based on the GCaMP3 reporter system by Leica fluorescence stereo microscopes (LFSM). LFSM combines super-fast manual screening with high-end imaging capabilities. Through this protocol, we can clearly observe the calcium flow after aphid’s piercing-sucking. Additionally, we describe a protocol to quantify Ca 2+ level using LFSM. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) .