Live imaging and quantitation of insect feeding-induced Ca(2+) signal using GCaMP3-based system in Nicotiana benthamiana

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).