Thin-film optical-acoustic combiner enables high-speed wide-field multi-parametric photoacoustic microscopy in reflection mode

Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of simultaneous high-resolution mapping of blood oxygenation and flow in vivo. However, its speed has been limited by the dense sampling required for blood flow quantification. To overcome this limitation, we have developed a high-speed multi-parametric PAM system, which enables simultaneous acquisition of ∼500 densely sampled B-scans by superposing the rapid optical scanning across the line-shaped focus of a cylindrically focused ultrasonic transducer over the conventional mechanical scan of the optical-acoustic dual foci. A novel, to the best of our knowledge, optical-acoustic combiner (OAC) is designed and implemented to accommodate the short working distance of the transducer, enabling convenient confocal alignment of the dual foci in reflection mode. A resonant galvanometer (GM) provides stabilized high-speed large-angle scanning. This new system can continuously monitor microvascular blood oxygenation (sO ) and flow over a 4.5 × 3 mm area in the awake mouse brain with high spatial and temporal resolutions (6.9 µm and 0.3 Hz, respectively).