We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. We demonstrate an imaging depth of 1,200 μm in vasculature and 1,160 μm in neurons. We also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.
Laser speckle contrast imaging (LSCI) is a powerful and simple method for full field imaging of blood flow. However, the depth dependence and the degree of multiple scattering have not been thoroughly investigated. We employ three-dimensional Monte Carlo simulations of photon propagation combined with high resolution vascular anatomy to investigate these two issues. We found that 95% of the detected signal comes from the top 700 μm of tissue. Additionally, we observed that single-intravascular scattering is an accurate description of photon sampling dynamics, but that regions of interest (ROIs) in areas free of obvious surface vessels had fewer intravascular scattering events than ROI over resolved surface vessels. Furthermore, we observed that the local vascular anatomy can strongly affect the depth dependence of LSCI. We performed simulations over a wide range of intravascular and extravascular scattering properties to confirm the applicability of these results to LSCI imaging over a wide range of visible and near-infrared wavelengths.