ADVANCES AND APPLICATIONS IN ULTRASOUND-SWITCHABLE FLUORESCENCE IMAGING

Abstract

**Please note that the full text is embargoed until 02/01/2026** Cancer diagnosis is a prerequisite for further medical therapy and treatment. Near-infrared (NIR) fluorescence imaging is a desirable modality without invasiveness and nonionizing radiation. However, fluorescence suffers from a strong light scattering in tissues, leading to a low spatial resolution and low sensitivity in centimeter-deep tissue (limited to several millimeters). To enhance spatial resolution and avoid the high scattering of conventional fluorescence imaging in deep tissue, ultrasound-switchable fluorescence (USF) imaging has emerged as a highly promising technique for biomedical imaging and research. The principle of improving spatial resolution in USF imaging is to confine the fluorescence emission from thermosensitive fluorophores to a small focal volume via focused ultrasound (FU) stimulations. As a result, the ambient background fluorescent signals can be effectively suppressed. The study achieved the size control of polymer-based and indocyanine green (ICG) encapsulated USF contrast agents, capable of accumulating in the tumor after intravenous injections. These nanoprobes varied in size from 58 nm to 321 nm. The bioimaging profiles demonstrated that the proposed nanoparticles can efficiently eliminate the background light from normal tissue and show a tumor-specific fluorescence enhancement in the BxPC-3 tumor-bearing mice models possibly via the enhanced permeability and retention effect. In vivo tumor USF imaging further demonstrated that these nanoprobes can effectively be switched 'ON' with enhanced fluorescence in response to a FU stimulation in the tumor microenvironment, contributing to the high-resolution USF images. Furthermore, USF technique was utilized to estimate the local background temperature for the first time by analyzing the temperature dependence of fluorescence emission from USF contrast agents induced by a FU beam. The USF contrast agent suspension was injected into a microtube that was embedded into a phantom and the dynamic USF signal was acquired using a camera-based USF system. The results showed that the difference between the temperatures acquired from the USF thermometry and the infrared thermography was 0.64 ± 0.43 °C when operating at the physiological temperature range from 35.27 to 39.31 °C. Lastly, an experimental investigation into the dynamics of interstitial fluid streaming and tissue recovery in animal tissue induced by the mechanical effects of acoustic waves was performed. Temperature-insensitive sulforhodamine-101 encapsulated poly(lactic-co-glycolic acid) nanoparticles with a size of 175 nm were locally injected into animal tissues. The changes of fluorescence over time caused by the streaming and backflow of interstitial fluid were studied with two ex vivo animal tissue models, and a faster recovery was observed in porcine tissue compared with the results in chicken tissue. In summary, in vivo USF imaging in tumor-bearing mouse models via intravenous (i.v.) injections of ICG-encapsulated poly (N-isopropylacrylamide) (PNIPAM) nanoparticles was achieved for the first time. Our findings suggest that ICG-PNIPAM agents are good candidates for USF imaging of tumors in live animals, indicating their great potential in optical tumor imaging in deep tissue. Except for imaging, the potential application of USF techniques in temperature measurements was also explored. The designed USF-based thermometry showed a broad application prospect in high spatial resolution temperature imaging with a tunable measurement range in deep tissue Finally, USF was employed to experimentally investigate the ultrasound-induced transport of nanoparticles in ex vivo tissues. This work has paved the way for the prospective integration of the USF imaging technique into various biomedical applications.