In Vivo Wide-Field Fluorescence Ca²⁺ Imaging of Xenopus Tadpole Brains

In vivo wide-field calcium ion (Ca²⁺) imaging enables visualization of neuronal activity across large brain regions in living Xenopus tadpoles. This approach provides a cost-effective and accessible alternative to two-photon microscopy for capturing population-level Ca²⁺ dynamics. The protocol outlines preparation of tadpoles expressing the genetically encoded Ca²⁺ indicator GCaMP6s, followed by cranial-window dissection, immobilization in low-melting-point agarose, and wide-field fluorescence imaging under low-intensity 480 nm excitation. Recordings are acquired at two frames per second for 30 min using a standard upright fluorescence microscope, and image sequences are processed in Fiji (ImageJ) to obtain ΔF/F₀ (%) traces from user-defined regions of interest. By maintaining intact brain physiology, this method enables reproducible detection of spontaneous or evoked neuronal activity in vivo. The workflow provides a practical framework for characterizing large-scale neural activity patterns and comparing experimental conditions across genetic, pharmacological, or developmental contexts.