One of the goals of systems neuroscience is to understand how sensory information is transformed into goal- directed behavior via diverse brain regions and circuits. To achieve this aim, it is critical to elucidate computations performed within specific layers of the cortex by specific cell classes and the communication dynamics between multiple brain regions. Two-photon microscopy has been used successfully to perform functional brain imaging at the single-cell level mice, but its penetration is limited by tissue scattering to the top layers of the cortex. I have developed a 3-photon microscope to overcome this challenge. Today, the main drawback of 3-photon microscope is its relatively modest speed, limiting its use for multi-site imaging. Optimizing instrument design and imaging protocol to overcome this limitation is required for broad end-user acceptance. In this proposal, I will construct and optimize a combined 2-photon and 3-photon microscope for multi-site, superficial and deep brain imaging at single-cell resolution. Specifically, I have first developed a custom-made 3-photon microscope with optimized laser and microscope parameters (Aim 1a). Optimizing these parameters can improve imaging speed and imaging depth while lowering the average laser power to avoid damage in the live mouse brain. The microscope performance improvement has been validated by performing functional imaging in the primary visual cortex of GCaMP6 mice to characterize visual responses of each cortical layer and subplate. In addition, I will characterize the effective attenuation lengths (EAL) of higher visual areas in awake mice with label-free imaging and laser-ablation methods. Then, I will demonstrate the microscope’s performance by examining cell-specific differences within a layer 6 (L6) of V1. Since neuronal responses to visual stimuli are modulated by the cortical state such as arousal, or reward expectation, I will image adjacent sets of neurons with distinct projections to the lateral geniculate nucleus (LGN) and lateral posterior (LP) regions (e.g., cortico-cortical [CC] and cortico-thalamic [CT] neurons in L6) in primary and higher visual areas to reveal circuit-based response types within a single cortical layer using retrobead-based tracing methods (Aim 1b). Next, I have developed custom-made 2-photon wide-field microscope to perform neuronal recordings and manipulations in the primary visual cortex and higher visual areas (Aim 2a). I have improved imaging speed and field of view by implementing multifocal multiphoton microscopy (MMM). Multiple foci two-photon excitation efficiency will be optimized by coupling a diffractive element (DOE) with customized intermediate optics. High sensitivity single-photon counting detection will be achieved using a novel avalanche photodiode array detector. To demonstrate microscope performance and which brain regions are necessary for a well-established goal-directed behavioral paradigm, I will perform SLM- ...