Contents 1. Introduction
Multiphoton microscopy enables high resolution, non-invasive optical imaging deep into scattering tissues such as the mouse brain. Using two low energy photons instead of one high energy photon to excite a fluorophore provides two major advantages. The first is that the lower energy, longer wavelength photons are scattered less. Thus more ballistic photons are able to reach the focus deep within the scattering sample. Second, the nonlinearly excited signal is strongly confined in space, with most of the signal generated within the focal volume. This spatial confinement breaks down when imaging at significant depths within scattering samples, since background fluorescence generated in the superficial regions (where the number of ballistic photons is the highest) eventually overwhelms the signal from the focus. For example, Theer et al used a regenerative amplifier to reach this limit in 2003. They used 225 nJ, 150 fs pulses at 925 nm to image GFP labeled neurons at a depth of 1-mm in the cortex of a mouse [1].
