Depth-compensated needle-shaped beam for in vivo photoacoustic microvascular imaging

Photoacoustic microscopy (PAM) exploits hemoglobin absorption in the visible spectrum for label-free microvascular imaging, but its shallow depth of field (DOF) limits in vivo applications. Existing DOF-extension strategies alleviate this constraint yet fail to correct tissue-induced optical attenuation, resulting in significant fluence loss at depth. Here, we present a PAM system (DC-DOE-PAM) integrated with a customized diffractive optical element (DC-DOE) that generates an axially modulated needle-shaped beam via wavefront engineering. The axial intensity modulation compensates for depth-dependent tissue attenuation, producing a uniform photon distribution over an extended focal range. The proposed PAM system employing a depth-compensated needle-shaped beam achieves a ninefold DOF extension (∼698 μm) while preserving capillary-level lateral resolution and consistent excitation fluence, enabling high-fidelity visualization of deep vasculature. In vivo experiments on mouse ear and brain demonstrate its ability to adapt to complex tissue environments and provide consistent vascular contrast across depths. Longitudinal monitoring of cerebral microcirculation further confirms stable high-resolution imaging and sensitivity to hemodynamic dynamics. This technique establishes a robust platform for quantitative vascular analysis and longitudinal studies of microvascular physiology.