Spatiospectral Photoacoustic Microscopy With Gain‐Switched Ti:Sapphire Laser for High‐Accuracy Molecular Dynamics Imaging

ABSTRACT Photoacoustic microscopy (PAM) enables high‐resolution, label‐free imaging by detecting ultrasound generated from pulsed‐laser absorption. For high‐fidelity spatiospectral imaging with single‐raster scanning, two spectrally distinct pulses should arrive nearly simultaneously to preserve spatial–temporal correlation, and at least one wavelength should be tunable for exogenous agents. We present spatiospectral PAM (SS‐PAM) driven by a gain‐switched Ti:sapphire laser engineered for this purpose. The source provides 735–855 nm tunability spanning hemoglobin and NIR‐I agents (e.g., indocyanine green, ICG) and is intrinsically synchronized to a 532 nm pump with a ≈150 ns gain‐switch delay, producing dual pulses for spatiospectral excitation. Operating at 20–30 kHz with pulse energies up to 63 µJ, SS‐PAM yields temporally correlated, high‐contrast excitation beyond sequential or dual‐laser schemes. In vivo, we quantified hemoglobin oxygen saturation (sO 2 ) under controlled hypoxia, tracked vascular transport after intravenous ICG, visualized peritumoral lymphatic drainage, and analyzed oxygen extraction in tumor‐associated microvasculature, establishing SS‐PAM as a robust platform for synchronized, vessel‐level functional and molecular imaging.