Vevo lazr system

RD@MBs at different concentrations (20, 40, 60, 80 and 100 μg/mL) were placed in agarose gel holes and US images in B-mode and Contrast mode were acquired with Vevo LAZR System (21 MHz, VisualSonics, Inc., Canada). For in vivo US imaging, tumor-bearing mice were intravenously injected with RD@MBs (3.5 mg/mL, 200 μL), and the US images of the tumor regions were acquired. The corresponding echo intensity was analyzed using an US imaging analysis software.
To invest drug delivery efficiency of UTMD technology, tumor-bearing mice were randomly divided into three groups including (1) free DiI, (2) DiI@MBs and DiI@MBs + US. The corresponding MBs (3.5 mg/mL, 200 μL) were intravenously administrated and the 3^rd group tumors were exposed to US irradiation immediately (1.0 MHz, 1.5 W/cm², 50% duty cycle, 3 min). Tumors were dissected and analyzed using FCM. A total of 10,000 events were collected for each sample.

Photoacoustic images were acquired using a Vevo LAZR system (VisualSonics Inc., Toronto, Canada) with excitation wavelengths from 680 nm to 970 nm, pulse duration 7-10ns, step size 1nm, frequency 20 Hz, and the 26mJ peak energy at the transducer end for all samples. The photoacoustic imaging signal was assessed by loading micelles at a concentration of 200 µM (based on the dye concentration) into polyethylene tubing (0.5 mm diameter). The tubes were placed in a plastic holder immersed in DI water. Images were acquired with the following settings: PA gains 40 dB, priority 99%, and distance 10 mm from the LZ250 transducer (75 µm axial resolution; 13-24 MHz broadband frequency).

The PA imaging performances of AuNPs and AuNS were evaluated in solidified 10% (w/v) gelatin phantoms (Figure S7a). 0.3 mL of AuNPs or AuNS solutions (200 µM gold, 2% (w/v) gelatin) was added into the sample holes on the phantom and solidified at 4 °C for 5 h. The VevoLAZR system (Visualsonics, Canada) was used to image the sample-bearing phantoms with excitation wavelengths from 680 to 970 nm. The PA stabilities of AuNPs and AuNS were performed in low-density polyethylene tubes (Figure S7b). Hence, 0.05 mL of AuNP or AuNS solutions (200 µM gold) were introduced in the polyethylene tubes and imaged with excitation wavelengths ranging from 680 to 970 nm. The PA intensities at 710 nm displayed in Figs. 4, 6 and 8 were determined from the spectral acquisition mode, which ranged from 680 to 970 nm. Only in the PA stability studies (Fig. 7), the PA intensities at 710 nm were obtained from single wavelength recordings, which were continuously acquired for 10 min.

Multiple blood vessel mimicking polymeric tubes were installed into Vevo®PHANTOM (Visualsonics, Fujifilm, Tokyo, Japan). ICG solutions mixed with distilled water and 20 mg/mL BSA were diluted to obtain eight different concentrations ranging from 0.125 to 5.0 mg/mL. These solutions were injected into the blood vessel mimicking tubes that were subjected to PAI in combination with conventional US (LZ-250, 20-MHz linear array transducer). Chicken breast muscle (6-mm-thick) which was purchased from grocery market was then placed over the tubes, the PA signal from ICG in the tubes was acquired in the range from 680 to 970 nm using a Vevo®LAZR system (Visualsonics, Fujifilm). To determine the maximum depth of signal penetration, 2.0 mg/mL of ICG in 50-uL tubes was placed at different depths. ranging from 0 to 22 mm deep)

Combined ex vivo US and PA imaging on mice legs was performed using the Vevo 2100 US device equipped with Vevo® LAZR system (VisualSonics, Amsterdam, The Netherlands) and the MX 250 transducer with a center frequency of 18 MHz. The BALB/c mouse cadavers were reused from a previous experiment, and, therefore, no live animals were needed or euthanized for the current study.
Prior to imaging, the mouse legs were shaved. Before intramuscular MB administration, control images were acquired using 4% acoustic power and a US gain of 8 dB for US NLC mode and a PA gain of 39 dB, wavelength range from 680 to 920 nm, for PA spectral imaging. The settings were adjusted to minimize non-specific signals that could come from the skin surface. After recording the control images, 1 × 10⁹ MB of selected samples were injected in each leg, followed by a 50 μL saline flush, and US and PA images were recorded at nearly similar transducer positions as for the control images and similar setup settings as for controls. For each sample, 4 mouse legs were imaged before and after MB injection.
In addition, a four times lower dose of ZnTTBNc encapsulated MB (2.5 × 10⁸ MB) was injected to evaluate the US and PA imaging capabilities at lower concentrations. US NLC and PA images were recorded following the same protocol as described before.

To investigate the performance of 3BP@PLGA-IR780 as a PA contrast agent, both in vitro and in vivo experiments were conducted. Firstly, 3BP@PLGA-IR780 suspension was stimulated by a PA laser with excitation wavelength ranging from 680 to 970 nm. Then, PA values of different concentrations of 3BP@PLGA-IR780 (the corresponding IR780 concentrations were 10, 20, 30, 40, 50 µg mL⁻¹) were measured, and corresponding PA images were attained. To evaluate the in vivo PA imaging performance, 4T1 tumor-bearing mice were intravenously injected with 200 μL of 3BP@PLGA-IR780 (PLGA equivalence: 10 mg mL⁻¹). The tumor PA images were collected at different time points (0, 1, 2, 4, 6, 24, and 48 h, n = 3) and the corresponding PA intensities were analyzed by a Vevo LAZR System (Visual Sonics Inc., Canada).