In Vivo Confocal Imaging of Fluorescently Labeled Microbubbles: Implications for Ultrasound Localization Microscopy.

We report the time kinetics of fluorescently labeled microbubbles (MBs) in capillary-level microvasculature as measured via confocal microscopy and compare these results to ultrasound localization microscopy (ULM). The observed 19.4 ± 4.2 MBs per confocal field-of-view ( [Formula: see text]) are in excellent agreement with the expected count of 19.1 MBs per frame. The estimated time to fully perfuse this capillary network was 193 s, which corroborates the values reported in the literature. We then modeled the capillary network as an empirically determined discrete-time Markov chain with adjustable MB transition probabilities though individual capillaries. The Monte Carlo random walk simulations found perfusion times ranging from 24.5 s for unbiased Markov chains up to 182 s for heterogeneous flow distributions. This pilot study confirms a probability-derived explanation for the long acquisition times required for super-resolution ULM.