Photoacoustic imaging (PAI) has great potential in the biomedical field. It best combines the high contrast of electromagnetic absorption for contrast agents and the high resolution of ultrasonic waves in biological tissues. However, limitations of endogenous contrast agents, such as nonspecific biodistribution, poor contrast in complex bio-environment, and few endogenous molecules absorbing above the visible region, will restrict the PAI applications. Then, PAI combined with exogenous contrast agents, which should have excellent stability, biocompatibility, and strong PA signal in deep tissue is required and will significantly promote PAI advance in different biomedical fields. In this thesis, we apply PAI into four different biomedical applications with suitable exogenous contrast agents, including biochemical substance imaging, microrobots visualization in the blood, tumor therapy guidance, and stem cell labeling and tracking which span from visible light, NIR-I window light to NIR-II window light excitation.

In Chapter 1, we first review the background of biomedical imaging. Then we briefly introduce the principle of PAI and main types of PAI instruments. Moreover, to better apply PAI into biomedical applications, six types of main exogenous contrast agents have been described. Last, we list some main PAI biomedical applications.

In Chapter 2, PAI has been used to measure H₂S concentration. First of all, a new activatable dual-modality probe has been developed absorbed at 532 nm, which combines with PAI as an H₂S sensing system. Then, excellent sensitivity and selectivity of the system have been measured in vitro, giving us a reference on in vivo experiments. Finally, based on cellular sensing results, H₂S sensing has been conducted on the subcutaneous model in vivo.

In Chapter 3, we show that PAI is a powerful tool to image microrobots in the blood. Firstly, the wavelength selection of PAI and the number of microrobots in blood circumstances have been investigated in which 800 nm was chosen to excite PAI. Moreover, depth imaging and movement of microrobots in the blood also has been demonstrated in vitro PAI experiments. At last, the movement of microrobots in the tail blood vessel and portal vein have been tracked by PAI and ultrasound imaging.

In Chapter 4, we employ PAI to guide deep glioma photothermal therapy (PTT). First, thiadiazoloquinoxaline-based semiconducting polymer nanoparticles (SPNs) has been introduced into near-infrared window II (NIR-II) PAI-guided glioma PTT theragnostic platform. Then, suitable particle size, excellent photostability, and deep PAI of the SPNs have been investigated in vitro, which indicated the feasibility of deep glioma therapy. More critical, PAI has been successfully used to monitor the peak accumulation of SPNs at tumor position after post-injection excited by 1064 nm, which has directly guided the PTT in vivo. Finally, efficient PAI-guided PTT toward gliomas under NIR-II light irradiation has been realized in both subcutaneously- and intracranially-implanted tumor models.

In Chapter 5, PAI has been applied in deep location labeling and long-term tracking of stem cells using 1064 nm. Firstly, new SPNs have been synthesized and processed by poly-L-lysine (PLL) to enhance the cellular uptake capability. Then, excellent photostability, good depth labeling, and cell uptake by SPNs have been demonstrated by PAI in vitro, exploring the feasibility of in vivo applications. Last, in vivo investigations reveal significant NIR-II PA contrast enhancement of the transplanted SPNs-labeled human mesenchymal stem cells by 40.6- and 21.7-fold in subcutaneous and brain imaging, respectively, compared with unlabeled cases.