Our understanding of the relationship between the structure and function of the intact brain is mainly shaped by magnetic resonance imaging. However, high resolution and deep-tissue imaging modalities are required to capture the subcellular relationship between structure and function, particularly in awake conditions. Here, we utilized a custom-made three-photon microscope to perform label-free third-harmonic generation (THG) microscopy as well as laser ablation to calculate effective attenuation lengths (EAL) of primary visual cortex and five adjacent visual cortical areas in awake mice. We identified each visual area precisely by retinotopic mapping via one-photon imaging of the calcium indicator GCaMP6s. EALs measured by depth-resolved THG microscopy in the cortex and white matter showed correspondence with the functional retinotopic sign map of each cortical area. To examine the basis for this correspondence, we used THG microscopy to examine several structural features of each visual area, including their cytoarchitecture, myeloarchitecture and blood vessel architecture. The cytoarchitecture of each area allowed us to estimate EAL values, which were comparable to experimental EAL values. The orientation of blood vessels and myelin fibers in the six areas were correlated with their EAL values. Ablation experiments, which provide ground truth measurements, generated 17 +- 3 % longer EALs compared to those obtained with THG imaging but were consistent with the latter. These results demonstrate a strong correlation between structural substrates of visual cortical areas, represented by EALs, and their functional visual field representation maps.
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