Label-Free Bond-Selective Imaging by Listening to Vibrationally Excited Molecules

We report the realization of vibrational photoacoustic (VPA) microscopy using optical excitation of molecular overtone vibration and acoustic detection of the resultant pressure transients. Our approach eliminates the tissue scattering problem encountered in near-infrared spectroscopy and enables depth-resolved signal collection. The 2nd overtone of the CH bond stretch around $8300\text{ }\text{ }{\mathrm{cm}}^{\ensuremath{-}1}$, where blood interference is minimal, is excited. We demonstrate 3D VPA imaging of lipid-rich atherosclerotic plaques by excitation from the artery lumen, and lipid storage in live Drosophila larvae, with millimeter-scale penetration depth.