The structure of brain regions is assumed to correlate with their function, but there are very few instances in which the relationship has been demonstrated in the live brain. This is due to the difficulty of simultaneously measuring functional and structural properties of brain areas, particularly at cellular resolution. Here, we performed label-free, third-harmonic generation (THG) microscopy to obtain a key structural signature of cortical areas, their effective attenuation lengths (EAL), in the vertical columns of functionally defined primary visual cortex and five adjacent visual areas in awake mice. EALs measured by THG microscopy in the cortex and white matter showed remarkable correspondence with the functional retinotopic sign map of each area. Structural features such as cytoarchitecture, myeloarchitecture and blood vessel architecture were correlated with areal EAL values, suggesting that EAL is a function of these structural features as an optical property of these areas. These results demonstrate for the first time a strong relationship between structural substrates of visual cortical areas and their functional representation maps in vivo. This study may also help in understanding the coupling between structure and function in other animal models as well as in humans.
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