Biomedical imaging techniques visualize the tissue structure and functional information, offering an important approach for disease study, diagnosis, and therapy. Photoacoustic imaging (PAI) is a new imaging technology that has been widely used in preclinical, physiological, and pathology studies. It can utilize both endogenous and exogenous absorption contrasts to image multiscale biological structures with high sensitivity as well as a high spatial resolution. Especially, optical-resolution photoacoustic microscopy (OR-PAM) is a noninvasive biomedical imaging technique that has a sub-cellular resolution, rich optical absorption contrast, and label-free imaging ability. OR-PAM shows its unique advantages in the resolution over photoacoustic computer tomography (PACT) which suffers from relatively low resolution despite better imaging depth. It can resolve fine vasculature and reveals the subtle alterations of blood vessels, which offers rich information for the study of various medical fields such as neuroscience, hemodynamic studies, etc. Unfortunately, the OR-PAM imaging speed is closely limited by the scanning method. The OR-PAM system parameters, such as sensitivity, scanning speed, large field of view (FOV), etc. are usually contradictory. It is significant to improve the performance of OR-PAM in these parameters. Here, we develop several kinds of OR-PAMs, expecting to achieve better performance in the sensitivity, imaging speed, FOV, and imaging contrast.

Firstly, we explore the system performance and determine the design method of OR-PAM. A typical OR-PAM probe is designed according to the pre-designed system parameters, showing an example of the designing of an OR-PAM probe. We explore the effect of the numerical aperture of the acoustic lens on the sensitivity. A high acoustic numeric aperture OR-PAM with enhanced sensitivity is developed to achieve hemoglobin imaging and functional imaging with laser pulse energy as low as 1 nJ. The basic structure of the mouse eye and brain can be imaged with the energy of 2 nJ.

To improve the imaging speed and FOV, we develop a wide-field polygon-scanning OR-PAM that for the first time achieves 1-MHz A-line rate with dual-wavelength. We address two technical challenges. The first is a 1-MHz dual-wavelength pulsed laser that has sufficient pulse energy and ultrafast switching time. The second is a polygon-scanning probe that has a high scanning speed, a large FOV, and excellent sensitivity. The OR-PAM system offers a B-scan rate of 477.5 Hz in a 12-mm range and a volumetric imaging rate of ~1 Hz over a 12×5 mm² scanning area. We image microvasculature and blood oxygen saturation in a 12×12 mm² scanning area in 5 s. Dynamic imaging of oxygen saturation in the mouse ear is demonstrated to monitor fast response to epinephrine injection. The new wide-field fast functional imaging ability broadens the biomedical application of OR-PAM.

Besides, multi-contrast OR-PAM can offer rich information. However, most OR-PAMs work on the single optical wavelength band, especially the visible region. Near-infrared-II window (NIR-II) light has attracted a lot of attention due to its good penetration depth and low background optical absorption. We develop multi-wavelength OR-PAM with a fiber-based visible/NIR PA probe. It can image tumor microvasculature, oxygen saturation, and nanoprobes in a single scanning. We develop a cost-efficient single laser source that provides 532, 558, and 1064-nm pulsed light with sub-micros wavelength switching time. Via dual-fiber illumination, we can focus the three beams to the same point. The optical and acoustic foci are confocally aligned to optimize the sensitivity. The visible and NIR wavelengths enable simultaneous tumor imaging with three different contrast modes. Results show obvious angiogenesis, significantly elevated oxygen saturation, and accumulated nanoparticles in the tumor regions, which offers comprehensive information to detect the tumor. This approach also allows us to identify feeding and draining vessels of the tumor and thus to determine local oxygen extraction fraction. In the tumor region, the oxygen extraction fraction significantly decreased along with tumor growth, which can also assist in tumor detection and staging. Fiber-based confocal visible/NIR photoacoustic microscopy offers a new tool for the early detection of cancer.

Finally, we report a fiber-based dual foci method that is employed to enhance the imaging speed of fast scanning OR-PAM without sacrificing the original sensitivity and FOV. A single-axis water-immersible resonant mirror is developed to achieve fast scanning with a B-scan rate of over 1000 Hz. It maintains the co-align of the laser beam and ultrasound beam thus maximizing the sensitivity in the FOV. Two 532-nm light beams are merged in the probe and focus adjacently on the sample. The proposed dual focus method can double the B-line rate to over 2000 Hz. We demonstrate the dual foci fast-scanning OR-PAM system by tracking the flowing ink with a C-scan rate of 3 Hz over 2×4 mm². We also monitor the drug response of a mice ear with a C-scan rate of 1.7 Hz in a large FOV. Experimental results show that the dual foci method enables the fast-scanning photoacoustic microscopy a new imaging tool in the study of fast physiological change.

In the future, the performance of these developed OR-PAMs can be continually improved. Multi-wavelength (more than two wavelengths) laser with a high pulse repetition ratio, can be configurated in the polygon scanner OR-PAM. It enables fast multi-parameter measurement in a large FOV. 1064 nm light can also be configurated in fast scanner OR-PAM. A fast scanning visible/NIR OR-PAM may be a powerful tool in clinical imaging. On the other hand, the integration of the multi-foci (more than two foci) method and fast scanner OR-PAM, such as voice coil scanner OR-PAM and polygon scanner OR-PAM with a large FOV, can achieve ultra-high-speed imaging without the sacrifice of the original performance of the fast scanner OR-PAM.

In conclusion, OR-PAM is a kind of high-performance imaging technique that can achieve non-invasive, label-free, multi-contrast, and high-resolution imaging. The developed OR-PAMs have improved imaging performance, especially in the imaging speed. Some experiments have been demonstrated to show the imaging capability of the developed OR-PAMs. These OR-PAMs are expected to offer new tools for biomedical imaging and broaden the application of OR-PAM.