Microbubble Timing and Vascular Geometry Are Associated With Vessel‐Level Blood–Brain Barrier Opening During Focused Ultrasound

Objectives To examine whether minute‐scale microbubble handling and injection‐to‐sonication timing are associated with vessel‐level focused ultrasound (FUS)–mediated blood–brain barrier opening (BBBO), and whether control normalization improves longitudinal 2‐photon (2P) measurements. Methods Cranial‐windowed mice received FITC–dextran (40 kDa) and SonoVue microbubbles by retro‐orbital injection, followed by 1.0 MHz FUS at 0.2–0.4 MPa. Two‐photon stacks were acquired over 0–15 minutes. Frangi vesselness and Otsu thresholding defined vessel masks and a perivascular ring for extravasation analysis. Three timing conditions were compared: standard (injection within ~5 seconds of reconstitution, sonication immediately); Microbubble Floating (microbubbles held 2 minutes before injection); and Sonication Delay (sonication started 2 minutes after injection). Per‐mouse AUC used 2‐way ANOVA; slice‐level vessel‐area dependence used a linear mixed‐effects model with mouse as a random intercept. Results Control intravascular fluorescence decreased by ~24% over 15 minutes, showing that the intravascular signal is not a stable denominator; control‐normalized extravascular AUC was therefore the primary metric. Under the standard protocol, extravascular AUC peaked at 0.3 MPa, but the across‐pressure difference was not significant ( F (2,6) = 3.36, p = .105). Timing effects were descriptive: relative to standard, both Microbubble Floating and Sonication Delay increased extravascular AUC at 0.2 MPa but decreased it at 0.3 MPa. Vessel area was inversely associated with extravasation at the slice level ( β = −0.37 per μm 2 , p = .010), with mouse‐level intraclass correlation (ICC = 0.80) indicating dominant between‐mouse variability. Conclusions Microbubble handling and injection‐to‐sonication timing are potential protocol variables in FUS‐BBBO studies. Control‐normalized extravascular metrics provide a reproducible framework for vessel‐level longitudinal 2‐photon quantification.