Two-photon imaging was developed partly as a response to these challenges (Denk et al., 1990). In this system, fluorophores are excited by absorption of a pair of photons each with wavelength double that of a photon capable of individually exciting a given fluorophore alone. Only at a very small (roughly 1 cubic micron) region surrounding the exact point of focus is light effectively absorbed to fully excite fluorescence. Thus, the non-linear absorption of light naturally provides axial resolution. This therefore limits photobleaching as well as any possible photodamage to points where the signal is acquired from. Two-photon imaging allows high spatial resolution imaging deep into tissues, including in vivo imaging in the mammalian brain (Denk et al., 1994), and also provides a means by which to carry out photochemical manipulations of cells and circuits (Denk, 1994).