Monte Carlo simulation of fluorescence imaging of microvasculature

Little numerical analysis has been done on in vivo vascular fluorescence imaging. Here, we use a 3D fluorescence Monte Carlo model to evaluate a microvasculature geometry obtained via two-photon microscopy. We found that a bulk-vascularization assumption does not pro- vide an accurate picture of penetration depth of the collected fluorescence signal. Instead the degree of absorption difference between extravascular and intravascular space, and the degree of stokes shift must be taken into account to determine the depth distribution. Additionally, we found that using targeted illumination can provide for superior surface vessel sensi- tivity over wide-field illumination, with small area detection offering an even greater amount of sensitivity to surface vasculature. Depth sensitivity can be enhanced by either increasing the detector area or increasing the illumination area. Finally, we see that excitation wavelength and vessel size can affect intra-vessel sampling distribution, as well as the amount of signal that originates from inside the vessel under targeted illumination conditions.