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The resolution of 3D Ultrasound Localization Microscopy (ULM) is determined by acquisition parameters such as frequency and transducer geometry but also by microbubble (MB) concentration, which is linked to the total acquisition time needed to sample the vascular tree at different scales. In this study, we introduce a novel 3D anatomically-realistic ULM simulation framework based on two-photon microscopy (2PM) and in-vivo MB perfusion dynamics. As a proof of concept, using metrics such as MB localization error, MB count and network filling, we quantify the effect of MB concentration and PSF volume by varying probe transmit frequency (3-15 MHz). We found that while low frequencies can achieve sub-wavelength resolution as predicted by theory, they are also associated with prolonged acquisition times to map smaller vessels, thus limiting effective resolution (i.e., the smallest vessel that can be reconstructed). A linear relationship was found between the maximal MB concentration and the inverse of the point spread function (PSF) volume. Since inverse PSF volume roughly scales cubically with frequency, the reconstruction of the equivalent of 10 minutes at 15 MHz would require hours at 3 MHz. We expect that these findings can be leveraged to achieve effective reconstruction and serve as a guide for choosing optimal MB concentrations in ULM.

An Anatomically Realistic Simulation Framework for 3D Ultrasound Localization Microscopy