Multi-wavelength photoacoustic microscopy enhanced by the high-sensitivity probe and reversible tissue transparent molecules

Abstract

Stimulated Raman scattering (SRS)-based multi-wavelength optical-resolution photoacoustic microscopy (MW-OR-PAM) enables high-speed functional imaging in vivo by providing rich molecular contrast. However, its broader biomedical application remains limited by the reduced hemoglobin absorption beyond 600 nm and the insufficient sensitivity of existing microscope probes. Here, we address these challenges through two key innovations. First, we develop a high-sensitivity acoustic-optical probe tailored for the SRS-based MW-OR-PAM system. The probe integrates a 9-μm-thick P(VDF-TrFE) film-based transducer between a planoconvex lens and a large-aperture acoustic lens, achieving a broad bandwidth (98.94%), a high numerical aperture (0.67), and adjustable optical focusing. Second, we introduce tartrazine, a biocompatible and tissue-transparent dye, to induce reversible optical clearing in vivo , thereby enhancing light penetration at wavelengths above 600 nm. These advances significantly improve microvascular imaging at longer wavelengths. We validate the performance of our system through high-resolution imaging of skin microvasculature, oxygen saturation mapping, and transcranial brain imaging. Our results establish MW-OR-PAM as a versatile and robust platform for functional and pathological imaging, effectively extending the spectral and penetration depth limitations of conventional OR-PAM systems in the red spectral region.