Diagram of the hardware of simultaneous Ca2+ imaging and 2pMAPG mapping. Two mode-locked femtosecond-pulse Ti:sapphire lasers set at 830 nm and 720 nm were connected to the laser-scanning microscope via two independent scanheads. The laser beam illumination times and diameters were regulated by acoustic optical modulators (AOMs) and collimating lens, respectively. A pyramidal cell in the cortical slice was patch-clamped and loaded with Alexa Fluor 594 and Fluo-5F. Extracellular solution containing caged glutamate was oxygenated and re-circulated continuously. On the PC screen, the Ca2+ imaging region (left) and 2pMAPG mapping area (right) were chosen. The imaging plane and mapping plane were changed rapidly by the piezo actuator attached to the objective. AOMs, Galvano mirrors, and the piezo actuator were regulated by FV1000-MPE software. Simultaneously, electric signals from the patch-clamp amplifier and from two photomultiplier tubes for detecting red and green fluorescence were recorded. If a photostimulated neuron innervated one of the spines in the imaging region, Ca2+ transients were observed in this spine (yellow in the PC screen). See the details in the main text.