1：Experimental Design and Method Selection:

The study employed microbubble-assisted focused ultrasound (FUS) to open the blood-brain barrier (BBB) and deliver silica-coated gold nanorods (Si-AuNRs) as contrast agents. Ultrasound-guided photoacoustic imaging (USPA) was used for visualization, with spectral unmixing techniques to distinguish exogenous from endogenous signals.

2：Sample Selection and Data Sources:

C57BL/6 mice were used as animal models. Samples included brain tissues from mice subjected to FUS BBB opening, with ex vivo and in vivo imaging performed.

3：List of Experimental Equipment and Materials:

Equipment included FUS transducers (2.25 MHz from Valpey Fisher, Inc. and 2 MHz from SonicConcepts, Inc.), function generator (Keysight Technologies, Inc.), RF amplifier (1040L, Electronics and Innovation, Inc.), 3D positioning system (Newport, Inc.), pulser/receiver (Olympus Co.), digital oscilloscope (Picoscope 3406D, Pico Technology), USPA imaging system (Vevo LAZR, VisualSonics, Inc.), UV-VIS spectrometer (Evolution 220, Thermo Scientific), two-photon microscope (LSM 710 NLO, Carl Zeiss AG). Materials included silica-coated gold nanorods (Nanohybrids, Inc.), microbubbles (Vevo MicroMarker, Visualsonics, Inc.), Evans Blue, isoflurane, oxygen, proparacaine, PBS, formalin, gelatin.

4：25 MHz from Valpey Fisher, Inc. and 2 MHz from SonicConcepts, Inc.), function generator (Keysight Technologies, Inc.), RF amplifier (1040L, Electronics and Innovation, Inc.), 3D positioning system (Newport, Inc.), pulser/receiver (Olympus Co.), digital oscilloscope (Picoscope 3406D, Pico Technology), USPA imaging system (Vevo LAZR, VisualSonics, Inc.), UV-VIS spectrometer (Evolution 220, Thermo Scientific), two-photon microscope (LSM 710 NLO, Carl Zeiss AG). Materials included silica-coated gold nanorods (Nanohybrids, Inc.), microbubbles (Vevo MicroMarker, Visualsonics, Inc.), Evans Blue, isoflurane, oxygen, proparacaine, PBS, formalin, gelatin. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Mice were anesthetized, positioned in a stereotax, and subjected to FUS sonication after co-injection of microbubbles and Si-AuNRs. USPA imaging was performed before and after sonication, with ex vivo imaging after euthanasia and perfusion. Histological analysis included H&E staining and two-photon microscopy.

5：Data Analysis Methods:

Image processing was done using MATLAB for spectral unmixing, including ratiometric analysis for ex vivo and advanced algorithms for in vivo imaging to resolve signals from AuNRs, deoxygenated hemoglobin, and oxygenated hemoglobin.