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 $8300{\mathrm{cm}}^{-1}$, 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.

Figure 1

Photoacoustic detection of molecules based on overtone absorption. (a) Schematic diagram of the 1st and 2nd overtone absorption of a molecule. (b) VPA imaging on an inverted microscope. T, ultrasound transducer; gray (red) beam, ns laser. (c) VPA spectra of biological molecules and tissue components. The VPA signal amplitude is normalized to the laser energy at each wavelength. The VPA peak around 1200 nm contains the contribution from 2nd overtone of symmetric and asymmetric stretching modes of ${\mathrm{CH}}_2$. The 3rd overtone band of the CH stretching can be observed around 920–935 nm.

Figure 2

Lipid depositions in atheromatous arteries mapped by VPA microscopy. (a) Schematic drawing of luminal inspection of an atheromatous artery by VPA microscopy. (b) VPA spectra elucidate progressively greater levels of lipid deposition inside an arterial wall at positions I, II, and III compared to a normal artery with no lipid deposition. (c)–(e) 3D reconstructed VPA images of a confluent lipid core in an atheromatous artery (c), a scattered lipid deposition in an arterial wall (d), and mild fatty streaks (e). 3D animations are available as supplementary videos; see 17.

Figure 3

In vivo 3D VPA imaging of fat bodies in a 3rd-instar larva of Drosophila melanogaster. (a) 3D reconstruction and maximum amplitude projections in $XZ$ and $XY$ planes, (b) transverse images, and (c) longitudinal (planar) images of the lipid storages in a Drosophila larva. (d) Depth-resolved VPA spectra at a location indicated by the light gray (yellow) arrow in (b). 3D animation is available as a supplementary video; see 17.