VASO fMRI is just one method of a large zoo of fMRI acquisition sequences that have been proposed for application of layer-fMRI, including: GE-BOLD, SE-BOLD, GRASE, ASL, etc. All of them (including VASO) are based on neurovascular coupling and can only capture fMRI signal changes in vascularized tissue. This means that, whenever a voxel has a higher vascularization and a higher microvascular density compared to other voxels, it will exhibit a higher fMRI responsiveness (a.k.a. vascular reactivity). As a result, layers with a higher vascular density are expected to be biased toward a larger fMRI signal change, for any task conditions. Thus, independent of the localization benefits of VASO compared to other methods, the layer-dependent microvascular density can introduce biases in layer-profiles. One extreme example would be layer I. Since layer I is almost free of microvascularization (Duvernoy 1981; Weber et al. 2008), none of the layer-fMRI sequences (including VASO) is expected to capture meaningful layer-specific functional signal from layer I. In other layers (II–VI), the VASO bias of layer-dependent vascular density is expected to be negligible for the majority
