2-photon calcium imaging is a powerful technique that is used to monitor the neural activity of individual neurons in the brain tissue in vivo using fluorescent activity sensors. The technique involves loading neurons with a fluorescent calcium-sensitive dyes. Changes in fluorescence signals indicate fluctuations in intracellular calcium, which is a reflection of spiking activity of the neurons. 2-photon calcium imaging is widely used by neuroscientists to study the activity of hundreds of individual neurons simultaneously. The advantages of two-photon microscopy include reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples.