Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of simultaneous, high-resolution mapping of blood hemoglobin concentration, 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 an ultra-high-speed multi-parametric PAM system, which enables simultaneous acquisition of ~500 densely sampled B-scans by superposing the rapid laser 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 optical-acoustic combiner is designed and implemented to accommodate the short working distance of the transducer, enabling convenient confocal alignment of the dual foci in the reflection mode. This new system enables continuous monitoring of microvascular hemoglobin concentration, blood oxygenation, and flow over a 4.5 x 3 mm2 area in the awake mouse brain with high spatial and temporal resolution (6.9 μm and 0.3 Hz, respectively).
High-speed photoacoustic microscopy of mouse cortical microhemodynamics