Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution

TLDR

Label‑free chemical imaging of biomolecules in living systems has been limited by water’s infrared absorption, low spatial resolution, and lack of optical sectioning. The study aims to overcome these limitations by detecting vibrational absorption–induced photothermal effects with a visible laser. The method uses a visible laser to capture the photothermal signal generated by mid‑infrared absorption. The mid‑infrared photothermal approach achieved 10 µM sensitivity, sub‑micrometer resolution, and enabled label‑free 3‑D chemical imaging of live cells and organisms, revealing lipid and drug distributions and permitting in vivo imaging of lipids and proteins.

Abstract

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption-induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in