Photoacoustic imaging (PAI) is an emerging biomedical imaging technique with a high contrast of optical imaging and high resolution and deep penetration of acoustic imaging, which has shown broad application prospects in the field of clinical disease diagnosis. An important implementation for realizing PAI is using acoustic-resolution photoacoustic microscopy (AR-PAM). Conventional light illumination methods have the problem of uneven light distribution, optical thermal noise, large energy loss, and decreased imaging sensitivity. Additionally, for the samples of complex biological tissues with irregular shapes, such as tumor and brain tissues, single-sided illumination methods have imaging limitations such as incomplete coverage of the target area and difficulty in obtaining accurate deep tissue information. In this paper, we report a dual-sided illumination method for AR-PAM. When compared with conventional methods, this method has higher imaging contrast in complex biological samples and can more accurately present the complete boundary of sample tissue. More comprehensive information was obtained, demonstrating the method’s promising potential in both clinical and preclinical research.
A polarization splitter was used in this study to divide the laser beam into two beams and they were coupled into the multimode optical fiber through fiber couplers. After being shaped with a planoconvex lens on both sides of an imaging probe, the emitted beams were irradiated to the imaging sample at a 45° angle. A high-frequency ultrasonic transducer received the photoacoustic signals generated by the sample. First, the feasibility of imaging was verified by creating two phantoms mimicking blood vessels at different depths. The imaging of popliteal lymph nodes, brain vasculature, and tumors in living mice with two illumination methods was then performed and compared, and their imaging performance with dual-sided illumination was more excellent than that with single-sided illumination method, proving the advantages of dual-sided illumination method in PAI of complex biological samples.
When the PAI results of the two tissue phantoms under different illumination schemes are compared, the overall signal-to-noise ratio and contrast of the images in the dual-sided illumination method are found to be better than those in the single-sided illumination method and more complete contour and depth information can be obtained for the imaging of complex samples (Fig. 3). In in vivo imaging experiments, the advantages of dual-sided illumination in improving imaging quality are also verified. Through imaging of indocyanine green traced (ICG-traced) mouse popliteal lymph nodes, the signal intensity of lymph nodes using dual-sided illumination method was approximately three times higher than that using the single-sided illumination method (Fig. 4). Noninvasive imaging of cerebral cortical blood vessels showed that the dual-sided illumination method can present more abundant microvessels in the marginal region with higher contrast (Fig. 5). Unlabeled in vivo imaging of mouse tumors was performed to evaluate the differences in peripheral vascular imaging between the two illumination methods, and the results showed that the blood vessels observed in the same area using the dual-sided illumination method were more abundant and tumor nourishing vessels were visible (Fig. 6). In addition, three-dimensional reconstruction of the tumor image showed that the dual-sided illumination method can image tumor edges more accurately and completely.
In this study, the imaging quality is improved by reconfiguring the light illumination method of AR-PAM from single-sided illumination to dual-sided illumination to achieve the homogeneous coverage of a laser beam for imaging complex biological tissues. The results show that the dual-sided illumination method improves contrast and signal-to-noise ratio for PAI in complex biological samples such as tissue phantoms, popliteal lymph nodes, brain vasculature, and tumors. Our study provides a new method for photoacoustic microscopy and has the potential to improve diagnosis accuracy in clinical and preclinical practices.
