Super-resolution functional photoacoustic microscopy via label-free cell tracking

Microvascular function and oxygen metabolism are central to tissue and organ health. However, label-free methods for imaging oxygen dynamics in three-dimensional (3D) microvascular networks at the level of single red blood cells (RBCs)-the fundamental units of oxygen transport in vivo-remain lacking. Here, we introduce super-resolution functional photoacoustic microscopy (SR-fPAM), which spatiotemporally tracks RBC movements under dual-wavelength excitation. SR-fPAM reconstructs super-resolved 3D microvascular architecture comparable to two-photon microscopy while providing quantitative measurements of RBC flow and oxygenation. In live mice, SR-fPAM revealed redistribution of oxygen and hemodynamics across 3D microvascular networks following a single-vessel stroke. These findings establish SR-fPAM as an enabling tool that bridges a critical gap in oxygen-metabolism imaging and opens new avenues for studying microvascular health and disease with unprecedented functional insights.