The overarching goal of this proposal is to push the depth penetration of multiphoton microscopy targeting neuroscience applications in need of large-scale recording of cortical activity where high resolution requirement (in the order of singl microns) cannot be relaxed.  To achieve deep high-resolution imaging while retaining sufficient signal-to-noise ratio of the measurements for imaging of activity (e.g., for detection of single spikes induced calcium transients), we will develop an unconventional non-degenerate 2-photon microscopy capitalizing on the recent practical demonstration of the advantage of using long wavelength light (~1700 nm) for deep penetration 3-photon microscopy but circumventing the low probability of 3-photon absorption (3PA).  Our deliverables - complementary to engineering efforts elsewhere aimed at large-volume sampling - would have a transformative impact on our ability to reconstruct spatially distributed neuronal circuit activity providing unprecedented opportunities for tests of biological hypotheses that are currently unfeasible.  The seed of the new technology is a well-known phenomenon where absorption of the second photon by the fluorophore molecule is enhanced through an intermediate state induced by absorption of the first photon.  This warrants an increase in the excitation efficiency given the right combination of the wavelengths.  For our goal of deep penetration, the IR beam will deliver high photon flux to the focal volume inside the cortical tissue.  The second higher energy photon beam will have lower intensity.  Thus, while the higher energy photon beam would experience higher scattering in the brain tissue, the flux requirement for this beam will be relaxed (compared to that in the conventional 2-photon microscopy) helping to achieve deep imaging.  Importantly, by increasing the intensity of the IR beam while lowering the intensity of the shorter wavelength beam, we will decrease the unwanted out-of-focus excitation on the brain surface.  This is because the shorter wavelength beam will not have enough photon density at the surface while the IR beam will lie outside the degenerate 2- photon absorption (2PA) range for visible emission fluorophores.  Finally, we will implement an innovative Adaptive Optics strategy to correct the phase distortions that will be experienced by the beam delivering higher energy photons.  Specifically, we use the IR beam, which can be focused well deep inside the tissue, as a reference point (guiding star) and adjusting the phase of the second beam to reach the maximum overlap.  Overall, we expect to achieve ~1.6 mm penetration inside the cortical tissue while avoiding excessive laser power and retaining the excitation volume characteristic for 2PA of the IR beam alone in the degenerate excitation mode.  Our endpoint deliverable will be a prototype device with the proof-of-concept demonstration of its performance for imaging of brain activity in vivo using synthetic calcium indicators and genetically encoded calcium-sensitive fluorescent proteins.  This project will lay the groundwork for an academic-industry partnership proposal in 3 years to fully develop and deploy the new technology as a commercial product.