Two-photon microscopy has had an enormous influence on animal studies of in vivo brain activity providing a tool for high-resolution imaging in live cortical tissue. Yet, the majority of 2-photon imaging studies, as of today, have focused on the top ~500 μm of the cerebral cortex due to limited penetration of light into biological tissues. Cerebral neurons, however, are wired in circuits spanning the entire cortical depth (~ 1 mm in mice), and sampling of activity throughout this depth would be required for reconstruction of circuit dynamics. Therefore, increasing the in vivo penetration depth of microscopic imaging is at the heart of brain initiative society. In this thesis, we demonstrate that the degree of freedom introduced by non-degenerate 2-photon excitation (ND-2PE), using two independently controlled pulsed laser sources of different photon energies, may provide a number of advantages over the conventional methods promising deeper penetration with higher efficiency of excitation and better signal-to-background ratio in a scattering medium. The presence of two laser beams allows:
1) Tuning the combination of wavelengths to optimize excitation cross-section for each fluorophore;
2) Independent control of power (and polarization) such that an increase in the IR power can be used to compensate for the loss of NIR power due to scattering and absorption;
3) Spatial displacement of the two beams to limit the overlap to the focal volume, minimizing background excitation;
All the advantages were validated with our experiments in this thesis. We started with investigating the cross section of widely used fluorophores under ND-2PE. The experimental results showed the enhancement to the cross-section of ND-2PE in comparison with that of D-2PE. An experiment for neuron images in mice brain also supported the signal enhancement under ND-2PE. Next, we demonstrated that ND-2PE can increase maximum excitation depth in a scattering phantom by compensating the loss of NIR beam with IR beam. Finally, we demonstrated that the out-out-focus excitation in scattering phantom can be reduced by strategically displacing two beams. The scheme of side-by-side beams was also applied to in vivo brain images.