Photoacoustic microscopy (PAM) is a hybrid imaging modality with high spatial resolution, excellent imaging contrast, moderate imaging depth, and functional imaging capabilities. Optical resolution photoacoustic microscopy (OR-PAM) can image blood oxygen saturation (sO₂) in vivo with excellent sensitivity and high resolution, providing an excellent tool for early cancer diagnosis and neurovascular research. In addition, real-time imaging of multi-functional parameters, such as total hemoglobin concentration (C_Hb) and sO₂, allows us to visualize real-time hemodynamic information in complex vascular networks, thereby enhancing our understanding of neurovascular and tissue metabolism studies. However, a single imaging method has certain limitations. Therefore, multimodal imaging systems are increasingly required to provide supplementary information to improve the specificity and sensitivity of disease diagnosis. The integrated PA/US microscopy can identify co-registered structural, functional, and molecular features with high resolution and sensitivity, making it an invaluable biomedical imaging tool. However, the main challenges we face are: (1) In OR-PAM, estimate the sO₂ measurement error caused by strong tissue scattering, and then recover more accurate sO₂ values. (2) In OR-PAM, accurately quantified dynamic changes of multi-functional parameters can be acquired at high imaging speed. (3) Integrate simultaneous dual-modal PA/US high-resolution imaging system. Therefore, the focus of my Ph.D. research is the development of High-speed dual-modal quantitative functional photoacoustic/ harmonic ultrasound microscopy.

The first part of my thesis discusses the simultaneous measurement of multi-functional parameters in vivo by single-shot multi-wavelength OR-PAM. The C_Hb can be obtained by measuring an isosbestic point of hemoglobin at a single wavelength (532 nm or 545 nm). For sO₂ measurements, conventional OR-PAM ignores wavelength-dependent light scattering in biological tissue. It considers that the PA amplitude can be approximated as a linear function of the absorption coefficient and obtains the in vivo sO₂ value by performing spectral unmixing at two wavelengths, such as 532 nm and 558 nm. However, strong, and non-uniform scattering can greatly attenuate the local light fluence even in shallow tissues. We propose a linearized wavelength-dependent fluence compensation algorithm to compensate for sO₂ calculation errors caused by strong tissue scattering. Monte Carlo simulations and phantom experiments show that the linear model can accurately estimate the local light fluence in the specified wavelength range (532 nm, 545 nm, 558 nm). Based on this conclusion, we constructed a three-wavelength (532 nm, 545 nm, and 558 nm) OR-PAM system based on the stimulated Raman scattering effect. The system uses PA signals at three wavelengths (532 nm, 545 nm, and 558 nm) to compensate for sO₂ measurement errors caused by strong tissue scattering. Functional brain imaging demonstrates that the compensation methods can improve sO₂ accuracy, especially in microvessels. Finally, we applied this new technique to study ischemic stroke. We monitored the whole process of ischemic stroke and reperfusion in mice. During this period, we monitored many important physiological parameters such as sO₂, oxygen extraction fraction (OEF), vessel density, arterial oxygen saturation (SaO₂), veinous oxygen saturation (SvO₂), etc. The results demonstrate self-fluence-compensated functional photoacoustic microscopy as a high-resolution functional imaging tool for studying stroke and other metabolic-related diseases.

The second part of my thesis focuses on the development of simultaneous dual-modal photoacoustic and harmonic ultrasound microscopy with an optimized acoustic combiner system. Simultaneous photoacoustic (PA) and ultrasound (US) imaging provide rich optical and acoustic contrast. However, due to the increased attenuation of high-frequency ultrasound, resolution and penetration depth are often at odds. To address this issue, we propose a simultaneous dual-modal PA/US microscopy with an optimized acoustic combiner that improves the penetration of ultrasound imaging while maintaining high resolution. Low-frequency ultrasonic transducers are used for acoustic transmission and high-frequency transducers are used for PA and US detection. The acoustic combiner is used to combine transmit and receive beams at a predetermined ratio. By combining two different transducers, harmonic ultrasound imaging, and high-frequency photoacoustic microscopy are realized. In vivo, experiments on the mouse brain demonstrated the simultaneous imaging capability of PA and US. Harmonic US imaging of mouse eyes reveals finer iris and lens boundary structures than conventional US imaging, providing a high-resolution anatomical reference for co-registered PA imaging. Subcutaneous tumor experiments allowed 3-D imaging of blood vessels and tumor borders, demonstrating the potential application of simultaneous dual-modal PA/US microscopy.

The third part of my thesis discusses the development of a high-speed buzzer-based functional photoacoustic microscopy system. First, the buzzer vibrates at its resonant frequency of 13 kHz, and the single-mode fiber on the buzzer will also vibrate. We use a voice coil motor for fast axis scanning and stepper motors for slow axis scanning. The high-speed OR-PAM system can achieve a 65 Hz B-can scan rate in the field of view (FOV) of 1 mm * 0.5 mm. In the field of view of 5 mm * 6 mm, the imaging time of the C-scan is 13 seconds, its imaging speed is nearly 40 times faster than the traditional OR-PAM, and its spatial resolution is the same as the traditional method. In vivo mouse ear imaging proves that the imaging system can restore photoacoustic images with high fidelity, and can even obtain microvascular structures, which also proves the high resolution of the system. Arteries and veins can be clearly distinguished from the sO₂ images, indicating that the system can monitor dynamic changes in function parameters in real-time.

In conclusion, during my Ph.D. research, I developed High-speed dual-modal quantitative functional PA/harmonic US microscopy, including self-fluence-compensated functional photoacoustic microscopy, simultaneous dual-modal PA/harmonic US microscopy with an optimized acoustic combiner, and high-speed buzzer-based functional OR-PAM. These technologies will actively promote the discovery of new potential applications of PA/US microscopy in various fields of basic life science and clinical practice.