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Intrinsic signal imaging (ISI) measures the hemodynamic response of the cortex to visual stimulation across the entire field of view. This "retinotopic map" effectively represents the spatial relationship of the visual field to locations within each cortical area. Retinotopic mapping was used to delineate functionally defined visual area boundaries, and enabled targeting of the in vivo two-photon calcium imaging to functionally defined locations in primary and secondary visual cortical areas.

Accurately mapping cortical areas involves first imaging the cortical surface with green LED illumination to capture fiduciary markers (vasculature landmarks). Next, imaging of the cortical surface during presentation of a visual stimulus with red LED illumination allows for capture of the hemodynamic response to visual stimuli.