Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow.

Capitalizing on the optical absorption of hemoglobin, photoacoustic microscopy (PAM) is uniquely capable of anatomical and functional characterization of the intact microcirculation in vivo. However, PAM of the metabolic rate of oxygen (MRO₂) at the microscopic level remains an unmet challenge, mainly due to the inability to simultaneously quantify microvascular diameter, oxygen saturation of hemoglobin (sO₂), and blood flow at the same spatial scale. To fill this technical gap, we have developed a multi-parametric PAM platform. By analyzing both the sO₂-encoded spectral dependence and the flow-induced temporal decorrelation of photoacoustic signals generated by the raster-scanned mouse ear vasculature, we demonstrated-for the first time-simultaneous wide-field PAM of all three parameters down to the capillary level in vivo.