Dual-channel high-speed functional photoacoustic microscopy with ultra-wide field of view

Photoacoustic microscopy (PAM) systems often face challenges in simultaneously achieving high speed, high resolution, high sensitivity, and a large field of view (FOV). To address this challenge, we have developed dual-channel PAM (DC-PAM) that can expand the FOV without compromising the imaging speed, detection sensitivity, or spatial resolution. DC-PAM has two identical, independent channels of laser excitation and acoustic detection. It exploits two facets of a single hexagon scanner to concurrently steer the dual excitation laser beams and the resultant acoustic waves. DC-PAM achieves an ultra-wide FOV of 22.5 × 24 mm² with a total functional imaging time of ~15 s. Proof-of-concept experiments were conducted using DC-PAM on freely-swimming zebrafish, hypoxia-challenged mice, and sleeping glassfrogs, all of which benefit from the large FOV and high imaging speed to track the dynamic and physiological processes at the whole-organ or whole-body level. These applications demonstrate the potential of DC-PAM for a wide range of biological studies.