Super-low-dose functional and molecular photoacoustic microscopy

Photoacoustic microscopy can image various biological molecules and nano-agents in vivo via low-scattering ultrasonic sensing. Insufficient sensitivity has been a long-standing obstacle for imaging of many low-absorbing chromophores with less photobleaching or toxicity, reduced perturbation to delicate organs, and more choices of lasers. Here, we present a multi-spectral super-low-dose photoacoustic microscopy (SLD-PAM) that is up to ~ 33-times more sensitive than the state of the art. We collaboratively innovate the photoacoustic probe design and implement a spectral-spatial filter. The new probe collects 300% more acoustic signals, and the filter further improves the signal-to-noise ratio by 200%~1100% across all optical wavelengths. SLD-PAM can visualize microvessels and quantify oxygen saturation (sO2) in vivo with ~ 1% of the maximum permissible exposure, dramatically reducing any possible phototoxicity or perturbation, especially in imaging of the eye and the brain. Capitalizing on the high sensitivity, we, for the first time, implement direct imaging of deoxyhemoglobin (Deoxy-Hb) concentration without spectral unmixing, avoiding wavelength-dependent and computational noises. We show that super-low-dose molecular imaging can reduce photobleaching by at least 85%. We also demonstrate SLD-PAM achieves similar molecular imaging quality using 80% fewer contrast agents, which greatly reduces potential biotoxicity. SLD-PAM enables the use of more low-absorbing nano-agents, small molecules, and genetically-encoded biomarkers, as well as more low-power light sources at wider spectra. We believe SLD-PAM offers a powerful tool for anatomical, functional, and molecular imaging.