It is well known that the brain tissue scatters light differently as a function of the wavelength. In general, shorter wavelengths scatter more and than longer wavelengths which means that longer wavelengths penetrate deeper in the tissue. Different studies have estimated the extent of brain volume that contribute to the hemodynamic signal under different wavelengths (480 nm to 630 nm) and concluded that most of the detected signal comes from depths up to ~1 mm with the more superficial tissue (~500 microns) contributing the most to the hemodynamic signal composition and deeper sections contributing progressively less. For instance, the mouse cortex is about ~1.5 mm deep with a vasculature comprised of superficial arteries and veins with branches that plunge into the cortex where a web of capillaries is present. So, based on the abovementioned properties of the tissue scattering, the intrinsic signal of the mouse cortex is mostly comprised of the hemodynamic signals of superficial blood vessels followed by the signals from capillaries up to layer V.

Due to its well-localized signal, the hemodynamic responses of capillaries have been utilized by numerous researchers interested in cortical mapping. In this context, the signals from superficial blood vessels may be somewhat undesirable. Given that the spectral properties of brain tissue are fixed, what strategies can be employed to attenuate the signals from superficial blood vessels?

To address this question, we evaluated the impact of focus depth and depth of field (DOF) on the quality of recorded intrinsic signals. This aims to provide guidance on adjusting these parameters to enhance the quality of cortical maps using optical imaging of intrinsic signals.